Interferon-like protein zcyto21

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for Zcyto21, an interferon-like protein, which is most closely related to interferon-α at the amino acid sequence level. The present invention also includes antibodies to the Zcyto21 polypeptides, and methods of using the polynucleotides and polypeptides.

[0001] The present application is a divisional of U.S. patentapplication Ser. No. 09/895,834, filed Jun. 29, 2001, which claims thebenefit of U.S. patent application Ser. No. 60/285,424, filed Apr. 20,2001, and U.S. patent application Ser. No. 60/215,446, filed Jun. 30,2000. U.S. patent application Ser. No. 09/895,834 is herein incorporatedby reference.

BACKGROUND OF THE INVENTION

[0002] Cellular differentiation of multicellular organisms is controlledby hormones and polypeptide growth factors. These diffusable moleculesallow cells to communicate with each other and act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

[0003] Hormones and growth factors influence cellular metabolism bybinding to receptor proteins. Certain receptors are integral membraneproteins that bind with the hormone or growth factor outside the cell,and that are linked to signaling pathways within the cell, such assecond messenger systems. Other classes of receptors are solubleintracellular molecules.

[0004] Cytokines generally stimulate proliferation or differentiation ofcells of the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.Cytokines play important roles in the regulation of hematopoiesis andimmune responses, and can influence lymphocyte development.

[0005] The human class II cytokine family includes interferon-α (IFN-α)subtypes, interferon-β (IFN-β), interferon-γ (IFN-γ), IL-10, IL-19 (U.S.Pat. No. 5,985,614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486,(1995)), IL-20 (Jiang et al., Oncogene 11, 2477-2486, (1995)), IL-22(Xie et al., J. Biol. Chem. 275, 31335-31339, (2000)), and AK-155(Knappe et al., J. Virol. 74, 3881-3887, (2000)). Most cytokines bindand transduce signals through either Class I or Class II cytokinereceptors. Members of human class II cytokine receptor family includeinterferon-αR1 (IFN-αR1), interferon-γ-R2 (IFN-γ-R2), interferon-γ R1(IFN-γ R1), interferon-γR2 (IFN-γR2), IL-10R (Liu et al., J. Immunol.152, 1821-1829, (1994)), CRF2-4 (Lutfalla et al. Genomics 16, 366-373,(1993)), IL-20Rβ (Blumberg et al., Cell 104, 9-19, (2001)) (also knownas zcytor7 (U.S. Pat. No. 5,945,511) and CRF2-8 (Kotenko et al.,Oncogene 19, 2557-2565, (2000)), IL-20Rβ (Blumberg et al., ibid, (2001))(also known as DIRS1 (PCT WO 99/46379)), IL-22RA1 (IL-22 receptor-α1,submitted to HUGO for approval) (also known as IL-22R (Xie et al., J.Biol. Chem. 275, 31335-31339, (2000)), zcytor11 (U.S. Pat. No.5,965,704) and CRF2-9 (Kotenko et al., Oncogene 19, 2557-2565, (2000)),and tissue factor.

[0006] Class II cytokine receptors are typically heterodimers composedof two distinct receptor chains, the α and β receptor subunits (Stahl etal., Cell 74, 587-590, (1993)). In general, the α subunits are theprimary cytokine binding proteins, and the β subunits are required forformation of high affinity binding sites, as well as for signaltransduction. An exception is the IL-20 receptor in which both subunitsare required for IL-20 binding (Blumberg et al., ibid, (2001)).

[0007] The class II cytokine receptors are identified by a conservedcytokine-binding domain of about 200 amino acids (D200) in theextracellular portion of the receptor. This cytokine-binding domain iscomprised of two fibronectin type III (FnIII) domains of approximately100 amino acids each (Bazan J. F. Proc. Natl. Acad. Sci. USA 87,6934-6938, (1990); Thoreau et al., FEBS Lett. 282, 16-31, (1991)). EachFnIII domain contains conserved Cys, Pro, and Trp residues thatdetermine a characteristic folding pattern of seven β-strands similar tothe constant domain of immunoglobulins (Uze et al., J. InterferonCytokine Res. 15, 3-26, (1995)). The conserved structural elements ofthe class II cytokine receptor family make it possible to identify newmembers of this family on the basis of primary amino acid sequencehomology. Previously we have successfully identified two new members ofclass II cytokine receptor family, zcytor7 (U.S. Pat. No. 5,945,511)(also known as IL-20R α (Blumberg et al., ibid, (2001)) and zcytor11(U.S. Pat. No. 5,965,704) (also known as IL-22R (Blumberg et al., ibid,(2001)), using this approach. Identification of additional novel membersof the class II cytokine receptor family is of interest becausecytokines play a vital role in regulating biological responses.

[0008] IL-22, also known as IL-TIF (IL-10-related T cell-derivedinducible factor) (Dumoutier et al., J. Immunology 164, 1814-1819,(2000)), is a recently described IL-10 homologue. Mouse IL-22 wasoriginally identified as a gene induced by IL-9 in T cells and mastcells in vitro (Dumoutier et al., J. Immunology 164, 1814-1819, (2000)).Acute phase reactant induction activity was observed in mouse liver uponIL-22 injection, and IL-22 expression was rapidly induced afterlipopolysaccharide (LPS) injection, suggesting that IL-22 contributes tothe inflammatory response in vivo (Dumoutier et al., Proc. Natl. Acad.Sci. U.S.A. 97, 10144-10149, (2000)).

[0009] The interleukins are a family of cytokines that mediateimmunological responses, including inflammation. The interleukinsmediate a variety of inflammatory pathologies. Central to an immuneresponse is the T cell, which produce many cytokines and adaptiveimmunity to antigens. Cytokines produced by the T cell have beenclassified as type 1 and type 2 (Kelso, A. Immun. Cell Biol. 76:300-317,1998). Type 1 cytokines include IL-2, IFN-γ, LT-α, and are involved ininflammatory responses, viral immunity, intracellular parasite immunityand allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-6,IL-10 and IL-13, and are involved in humoral responses, helminthimmunity and allergic response. Shared cytokines between Type 1 and 2include IL-3, GM-CSF and TNF-α. There is some evidence to suggest thatType 1 and Type 2 producing T cell populations preferentially migrateinto different types of inflamed tissue.

[0010] Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3^(rd)Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), andby Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages158-188 (John Wiley & Sons 1998)). Interferons exhibit a variety ofbiological activities, and are useful for the treatment of certainautoimmune diseases, particular cancers, and the enhancement of theimmune response against infectious agents, including viruses, bacteria,fungi, and protozoa. To date, six forms of interferon have beenidentified, which have been classified into two major groups. Theso-called “type I” interferons include interferon-α, interferon-β,interferon-107 , interferon-δ, and interferon-τ. Currently, interferon-γand one subclass of interferon-α are the only type II interferons.

[0011] Type I interferons, which are thought to be derived from the sameancestral gene, have retained sufficient similar structure to act by thesame cell surface receptor. The α-chain of the human interferon-α/βreceptor comprises an extracellular N-terminal domain, which has thecharacteristics of a class II cytokine receptor. Interferon-γ does notshare significant homology with the type I interferons or with the typeII interferon-α subtype, but shares a number of biological activitieswith the type I interferons.

[0012] In humans, at least 16 non-allelic genes code for differentsubtypes of interferon-α, while interferons β and ω are encoded bysingle genes. Type I interferon genes are clustered in the short arm ofchromosome 9. Unlike typical structural human genes, interferon-α,interferon-β, and interferon-ω lack introns. A single gene for humaninterferon-γ is localized on chromosome 12 and contains three introns.To date, interferon-τ has been described only in cattle and sheep, whileinterferon-δ has been described only in pigs.

[0013] Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of interferon-α has been approved for use in morethan 50 countries for the treatment of medical conditions such as hairycell leukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of interferon-β totreat multiple sclerosis, a chronic disease of the nervous system.Interferon-γ is used to treat chronic granulomatous diseases, in whichthe interferon enhances the patient's immune response to destroyinfectious bacterial, fungal, and protozoal pathogens. Clinical studiesalso indicate that interferon-γ may be useful in the treatment of AIDS,leishmaniasis, and lepromatous leprosy.

[0014] The demonstrated in vivo activities of the cytokine familyillustrate the enormous clinical potential of, and need for, othercytokines, cytokine agonists, and cytokine antagonists. The presentinvention addresses these needs by providing a new cytokine thatstimulates cells of the hematopoietic cell lineage, as well as relatedcompositions and methods.

BRIEF DESCRIPTION OF THE FIGURES

[0015] The FIGURE is a Hopp/Woods hydrophilicity profile of the ZZcyto21protein sequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the FIGURE bylower case letters.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0017] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0018] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0019] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0020] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0021] The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0022] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0023] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0024] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0025] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0026] The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

[0027] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0028] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0029] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0030] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

[0031] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0032] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0033] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0034] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6receptor).

[0035] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0036] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0037] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0038] All references cited herein are incorporated by reference intheir entirety.

[0039] Zcyto21 gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:2. The signal sequence for Zcyto21 can be predicted ascomprising amino acid residue 1 (Met) through amino acid residue 19(Ala) of SEQ ID NO:2. The mature peptide for Zcyto21 begins at aminoacid residue 20 (Gly).

[0040] The Zcyto21 gene is contained in BAC sequences AC011445, andAC018477 which have been mapped to human chromosome 19q13.13. Thisregion of chromosome 19 may also comprise a cluster of interferon-likegenes. A consensus cDNA showing a polynucleotide sequence of Zcyto21 isshown in SEQ ID NO:6 and the polypeptide it encodes is shown in SEQ IDNO:7.

[0041] As described below, the present invention provides isolatedpolypeptides having an amino acid sequence that is at least 70%, atleast 80%, or at least 90%, 95%, 96%, 97%, 98% or 99% identical toeither amino acid residues 20 to 200 of SEQ ID NO:2 or amino acidresidues 1 to 200 of SEQ ID NO:2. The present invention also providesisolated polypeptides having an amino acid sequence that is at least70%, at least 80%, or at least 90%, 95%, 96%, 97%, 98% or 99% identicalto either amino acid residues 20 to 219 of SEQ ID NO:9 or amino acidresidues 1 to 219 of SEQ ID NO:9. The present invention also providesisolated polypeptides having an amino acid sequence that is at least70%, at least 80%, or at least 90%, 95%, 96%, 97%, 98% or 99% identicalto either amino acid residues 20 to 203 of SEQ ID NO:12 or amino acidresidues 1 to 203 of SEQ ID NO:12. The present invention also includes apolypeptide that further comprises a signal secretory sequence thatresides in an amino-terminal position relative to the first amino acidsequence, wherein the signal secretory sequence comprises amino acidresidues 1 to 19 of the amino acid sequence of SEQ ID NO:2.

[0042] In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions, and are more highly conserved amongmembers of the family. However, the interferons (INF), andinterferon-alpha and interferon-tau in particular, are characterized assix helix bundles. INF helix A is equivalent to helix A of Zcyto21; INFhelix B is equivalent to helix C of Zcyto21; INF helix C is equivalentto helix D of Zcyto21, and INF helix D is equivalent to helix F ofZcyto21. Thus, the loop between the AB loop, and CD loop of INF isexpanded in Zcyto21 to contain short helices B and E of Zcyto21.

[0043] Zcyto21 helices are predicted as follows: helix A is defined byamino acid residues 49 (Ser) to 63 (Leu); helix B by amino acid residues76 (Asn) to 84(Val); helix C by amino acid residues 89 (Val) to 104(Ala); helix D by amino acid residues 111 (Glu) to 133 (Gln); helix E byamino acid residues 137 (Thr) to 158 (Lys); and helix F by amino acidresidues 163 (Gly) to 189 (Leu); as shown in SEQ ID NO: 2. The cysteineresidues are conserved between Zcyto21, and INF-α, and may form anintermolecular disulfide bond, in particular to form homodimers withadditional Zcyto21 molecules. Further analysis of Zcyto21 based onmultiple alignments predicts that cysteines at amino acid residues 34and 131, and 68 and 164, (as shown in SEQ ID NO: 2) will formintramolecular disulfide bonds. The cysteine at residue 190 is free, andmay form an intermolecular disulfide association. The correspondingpolynucleotides encoding the Zcyto21 polypeptide regions, domains,motifs, residues and sequences described herein are as shown in SEQ IDNO:1. The degenerate polynucleotide sequence of SEQ ID NO:2 is shown inSEQ ID NO:3. The degenerate polynucleotide sequence of SEQ ID NO:9 isshown in SEQ ID NO:10. The degenerate polynucleotide sequence of SEQ IDNO:12 is shown in SEQ ID NO:13.

[0044] Detailed mutational analysis of murine IL-2 (Zurawski et al.,EMBO J. 12:5113-5119, 1993) shows residues in helices A and C areimportant for binding to IL-2Rβ; critical residues are Asp₃₄, Asn₉₉, andAsn₁₀₃. Multiple residues within murine IL-2 loop A/B and helix B areimportant for IL-2Rα binding, while only a single residue, Gln141 inhelix D, is vital for binding with IL-2Rα. Similarly, helices A and Care sites of interaction between IL-4 and IL-4Rα (the structurallysimilar to IL-2Rα), and residues within helix D are vital for IL-2Rαinteraction (Wang et al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997;Kruse et al., EMBO J. 11:3237-3244, 1992). In particular, the mutationTyr₁₂₄ to Asp in human IL-4 creates an antagonist, which binds withIL-4Rα but not IL-2Rα and therefore cannot signal (Kruse et al. ibid.1992).

[0045] Four-helical bundle cytokines are also grouped by the length oftheir component helices. “Long-helix” form cytokines generally consistof between 24-30 residue helices, and include IL-6, ciliaryneutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and humangrowth hormone (hGH). “Short-helix” form cytokines generally consist ofbetween 18-21 residue helices and include IL-2, IL-4 and GM-CSF. Studiesusing CNTF and IL-6 demonstrated that a CNTF helix can be exchanged forthe equivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines are determined on the basis of structural homology,irrespective of sequence identity, and can maintain functional integrityin a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).Therefore, the helical domains of Zcyto21 will be useful for preparingchimeric fusion molecules, particularly with other interferons todetermine and modulate receptor binding specificity. Of particularinterest are fusion proteins that combine helical and loop domains frominterferons and cytokines such as INF-α, IL-10, human growth hormone.

[0046] Zcyto21 mRNA has been identified in tissues of brain, islet,prostate, testis, pituitary, placenta, ovarian tumor, lung tumor, rectaltumor and ovarian tumor, as well as an activated immune cell line (CD3+)and a prostate epithelial cell line, which had been transformed withhuman papilloma virus IV (HPVS).

[0047] The present invention provides polynucleotide molecules,including DNA and RNA molecules, that encode the Zcyto21 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ IDNOS:3, 10, and 13 are degenerate DNA sequences that encompasses all DNAsthat encode the Zcyto21 polypeptide of SEQ ID NOS:2, 9, and 12,respectively. Those skilled in the art will recognize that thedegenerate sequence of SEQ ID NO:3, for example, also provides all RNAsequences encoding SEQ ID NO:2 by substituting U for T. Thus, Zcyto21polypeptide-encoding polynucleotides comprising nucleotide 1 or 58 tonucleotide 603 of SEQ ID NO:3 and their RNA equivalents are contemplatedby the present invention. Table 1 sets forth the one-letter codes usedwithin SEQ ID NO:3 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, withA being complementary to T, and G being complementary to C. TABLE 1Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T TA A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T WA|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T NA|C|G|T N A|C|G|T

[0048] The degenerate codons used in SEQ ID NOS:3, 10 and 13,encompassing all possible codons for a given amino acid, are set forthin Table 2. TABLE 2 One Amino Letter Degenerate Acid Code Codons CodonCys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACGACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGAGGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GARGln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGNLys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTGCTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TACTAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SARAny X NNN

[0049] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0050] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 3). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0051] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of Zcyto21RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), or by screening conditioned medium from various celltypes for activity on target cells or tissue. Once the activity or RNAproducing cell or tissue is identified, total RNA can be prepared usingguanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding Zcyto21 polypeptides are then identified andisolated by, for example, hybridization or PCR.

[0052] A longer clone encoding Zcyto21 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to Zcyto21 receptor fragments, or other specificbinding partners.

[0053] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are Zcyto21 polypeptides from other mammalianspecies, including murine, porcine, ovine, bovine, canine, feline,equine, and other primate polypeptides. Orthologs of human Zcyto21 canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses Zcyto21 as disclosed herein. Suitable sources ofmRNA can be identified by probing Northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared frommRNA of a positive tissue or cell line. A Zcyto21-encoding cDNA can thenbe isolated by a variety of methods, such as by probing with a completeor partial human cDNA or with one or more sets of degenerate probesbased on the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human Zcyto21 sequencedisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to Zcyto21 polypeptide,binding studies or activity assays. Similar techniques can also beapplied to the isolation of genomic clones.

[0054] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human Zcyto21band that allelic variation and alternative splicing are expected tooccur. Allelic variants of this sequence can be cloned by probing cDNAor genomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the Zcyto21 polypeptide, are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art. Examplesof alternatively spliced variants are shown in SEQ ID NO:8 (SEQ ID NO:9for the corresponding polypeptide), and in SEQ ID NO:11 (SEQ ID NO:12for the corresponding polypeptide). An example of an allelic variant isshown in SEQ ID NO:4, which corresponds to the polypeptide sequence asshown in SEQ ID NO:5. There is a polymorphism between the polypeptidesequence as shown in SEQ ID NO:1 and that shown in SEQ ID NO:4 atnucleotide number 572. This polymorphism might create an antagonist ofZcyto21 or a molecule of reduced or altered function, which might leadto a higher likelihood of disease susceptibility.

[0055] The present invention also provides reagents, which will find usein diagnostic applications. For example, the Zcyto21 gene, a probecomprising Zcyto21 DNA or RNA or a subsequence thereof can be used todetermine if the Zcyto21 gene is present on a human chromosome, such aschromosome 19, or if a gene mutation has occurred. Zcyto21 is located atthe q13.13 region of chromosome 19. Detectable chromosomal aberrationsat the Zcyto21 gene locus include, but are not limited to, aneuploidy,gene copy number changes, loss of heterogeneity (LOH), translocations,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995).

[0056] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0057] For example, Delague et al., (Am. J. Hum. Genet. 67: 236-243,2000) identified that Charcot-Marie-Tooth disease is localized to19q13.1-13.3 (Delague et al., Am. J. Hum. Genet. 67: 236-243, 2000).

[0058] A diagnostic could assist physicians in determining the type ofdisease and appropriate associated therapy, or assistance in geneticcounseling. As such, the inventive anti-Zcyto21 antibodies,polynucleotides, and polypeptides can be used for the detection ofZcyto21 polypeptide, mRNA or anti-Zcyto21 antibodies, thus serving asmarkers and can be directly used for detecting or genetic diseases orcancers, as described herein, using methods known in the art anddescribed herein. Further, Zcyto21 polynucleotide probes can be used todetect abnormalities or genotypes associated with chromosome 19deletions and translocations associated with human diseases or othertranslocations involved with malignant progression of tumors or other19q13.13 mutations, which are expected to be involved in chromosomerearrangements in malignancy; or in other cancers. Similarly, Zcyto21polynucleotide probes can be used to detect abnormalities or, genotypesassociated with chromosome 19q13.13 trisomy and chromosome lossassociated with human diseases or spontaneous abortion. Thus, Zcyto21polynucleotide probes can be used to detect abnormalities or genotypesassociated with these defects.

[0059] In general, the diagnostic methods used in genetic linkageanalysis, to detect a genetic abnormality or aberration in a patient,are known in the art. Analytical probes will be generally at least 20 ntin length, although somewhat shorter probes can be used (e.g., 14-17nt). PCR primers are at least 5 nt in length, preferably 15 or more,more preferably 20-30 nt. For gross analysis of genes, or chromosomalDNA, a Zcyto21 polynucleotide probe may comprise an entire exon or more.Exons are readily determined by one of skill in the art by comparingZcyto21 sequences (SEQ ID NO:1) with the genomic DNA for Zcyto21. Ingeneral, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Most diagnostic methods comprise the steps of (a) obtaining agenetic sample from a potentially diseased patient, diseased patient orpotential non-diseased carrier of a recessive disease allele; (b)producing a first reaction product by incubating the genetic sample witha Zcyto21 polynucleotide probe wherein the polynucleotide will hybridizeto complementary polynucleotide sequence, such as in RFLP analysis or byincubating the genetic sample with sense and antisense primers in a PCRreaction under appropriate PCR reaction conditions; (iii) Visualizingthe first reaction product by gel electrophoresis and/or other knownmethod such as visualizing the first reaction product with a Zcyto21polynucleotide probe wherein the polynucleotide will hybridize to thecomplementary polynucleotide sequence of the first reaction; and (iv)comparing the visualized first reaction product to a second controlreaction product of a genetic sample from wild type patient. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the diseased orpotentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the Zcyto21 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, thecomplement of SEQ ID NO:1, or an RNA equivalent thereof. Such methods ofshowing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics seesee, generally, Mathew (ed.), Protocols in Human Molecular Genetics(Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methodsand Applications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

[0060] Mutations associated with the Zcyto21 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an Zcyto21 gene for a mutation can beperformed using a subject's genomic DNA. Methods for amplifying genomicDNA, obtained for example from peripheral blood lymphocytes, arewell-known to those of skill in the art (see, for example, Dracopoli etal. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7(John Wiley & Sons 1998)).

[0061] Within embodiments of the invention, isolated Zcyto21-encodingnucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules having the nucleotide sequence of SEQ ID NO:1, tonucleic acid molecules having the nucleotide sequence of nucleotides 58to 603 of SEQ ID NO:1, or to nucleic acid molecules having a nucleotidesequence complementary to SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

[0062] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA andDNA-RNA, can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

[0063] It is well within the abilities of one skilled in the art toadapt these conditions for use with a particular polynucleotide hybrid.The T_(m) for a specific target sequence is the temperature (underdefined conditions) at which 50% of the target sequence will hybridizeto a perfectly matched probe sequence. Those conditions which influencethe T_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

[0064] Following hybridization, the nucleic acid molecules can be washedto remove non-hybridized nucleic acid molecules under stringentconditions, or under highly stringent conditions. Typical stringentwashing conditions include washing in a solution of 0.5×-2×SSC with 0.1%sodium dodecyl sulfate (SDS) at 55-65° C. That is, nucleic acidmolecules encoding a variant Zcyto21 polypeptide hybridize with anucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (orits complement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

[0065] Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantZcyto21 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

[0066] The present invention also provides isolated Zcyto21 polypeptidesthat have a substantially similar sequence identity to the polypeptidesof SEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, or greater than95%, 96%, 97%, 98%, or 99% sequence identity to the sequences shown inSEQ ID NO:2, or their orthologs. The present invention also includespolypeptides that comprise an amino acid sequence having at least 70%,at least 80%, at least 90%, at least 95% or greater than 95%, 96%, 97%,98%, or 99% sequence identity to the sequence of amino acid residues 1to 200 or 20 to 200 of SEQ ID NO:2. The present invention furtherincludes nucleic acid molecules that encode such polypeptides. Methodsfor determining percent identity are described below.

[0067] The present invention also contemplates variant Zcyto21 nucleicacid molecules that can be identified using two criteria: adetermination of the similarity between the encoded polypeptide with theamino acid sequence of SEQ ID NO:2, and/or a hybridization assay, asdescribed above. Such Zcyto21 variants include nucleic acid molecules:(1) that hybridize with a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 (or its complement) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C.; or (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%,96%, 97%, 98%, or 99% sequence identity to the amino acid sequence ofSEQ ID NO:2. Alternatively, Zcyto21 variants can be characterized asnucleic acid molecules: (1) that hybridize with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 (or its complement) underhighly stringent washing conditions, in which the wash stringency isequivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) thatencode a polypeptide having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:2.

[0068] Percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V A  4R −1  5 N −2  0  6 D −2 −2  1  6 C  0  3 −3 −3  9 Q −1  1  0  0 −3  5 E−1  0  0  2 −4  2  5 G  0 −2  0 −1 −3 −2 −2  6 H−2  0  1 −1 −3  0  0 −2  8 I −1 −3 −3 −3 −1 −3 −3 −4 −3  4 L−1 −2 −3 −4 −1 −2 −3 −4 −3  2  4 K −1  2  0 −1 −3  1  1 −2 −1 −3 −2  5 M−1 −1 −2 −3 −1  0 −2 −3 −2  1  2 −1  5 F−2 −3 −3 −3 −2 −3 −3 −3 −1  0  0 −3  0  6 P−1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4  7 S 1 −1  1  0 −1  0  0  0 −1 −2 −2  0 −1 −2 −1  4 T 0 −1  0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1  1  5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1  1 −4 −3 −2 11 Y−2 −2 −2 −3 −2 −1 −2 −3  2 −1 −1 −2 −1  3 −3 −2 −2  2  7 Z 0 −3 −3 −3 −1 −2 −2 −3 −3  3  1 −2  1 −1 −2 −2  0 −3 −1  4

[0069] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant Zcyto21. The FASTA algorithm is described by Pearsonand Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson,Meth. Enzymol. 183:63 (1990).

[0070] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0071] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as default.

[0072] Variant Zcyto21 polypeptides or polypeptides with substantiallysimilar sequence identity are characterized as having one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions (seeTable 4) and other substitutions that do not significantly affect thefolding or activity of the polypeptide; small deletions, typically ofone to about 30 amino acids; and amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from about 149 to 230 amino acid residuesthat comprise a sequence that is at least 70%, preferably at least 90%,and more preferably 95%, 96%, 97%, 98%, 99% or more identical to thecorresponding region of SEQ ID NO:2. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between theZcyto21 polypeptide and the affinity tag. Preferred such sites includethrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0073] Determination of amino acid residues that comprise regions ordomains that are critical to maintaining structural integrity can bedetermined. Within these regions one can determine specific residuesthat will be more or less tolerant of change and maintain the overalltertiary structure of the molecule. Methods for analyzing sequencestructure include, but are not limited to alignment of multiplesequences with high amino acid or nucleotide identity, secondarystructure propensities, binary patterns, complementary packing andburied polar interactions (Barton, Current Opin. Struct. Biol.5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10,1996). In general, when designing modifications to molecules oridentifying specific fragments determination of structure will beaccompanied by evaluating activity of modified molecules.

[0074] Amino acid sequence changes are made in Zcyto21 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, where the Zcyto21 polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners. The effects ofamino acid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

[0075] A Hopp/Woods hydrophilicity profile of the Zcyto21 proteinsequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc.Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. For example, in Zcyto21,hydrophilic regions include residues 155 (Glu) to 160 (Glu); residues 51(Lys) to 56 (Ala); residues 50 (Phe) to 55 (Asp); residues 140 (Pro) to145 (Arg); and residues 154 (Gln) to 159 (Lys); as shown in SEQ ID NO:2.

[0076] Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a Zcyto21 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp.

[0077] The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between INF-α and other interferons.Using methods such as “FASTA” analysis described previously, regions ofhigh similarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant Zcyto21 polynucleotide on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcyto21 gene can hybridize to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, as discussed above.

[0078] Other methods of identifying essential amino acids in thepolypeptides of the present invention are procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl.Acad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological or biochemical activity as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699 (1996).

[0079] The present invention also includes functional fragments ofZcyto21 polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” Zcyto21 or fragment thereof as defined hereinis characterized by its proliferative or differentiating activity, byits ability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-Zcyto21 antibody or Zcyto21receptor (either soluble or immobilized). As previously describedherein, Zcyto21 is characterized by a six-helical-bundle structurecomprising: helix A is defined by amino acid residues 49 (Ser) to 63(Leu); helix B by amino acid residues 76 (Asn) to 84(Val); helix C byamino acid residues 89 (Val) to 104 (Ala); helix D by amino acidresidues 111 (Glu) to 133 (Gln); helix E by amino acid residues 137(Thr) to 158 (Lys); and helix F by amino acid residues 163 (Gly) to 189(Leu); as shown in SEQ ID NO: 2. Thus, the present invention furtherprovides fusion proteins encompassing: (a) polypeptide moleculescomprising one or more of the helices described above; and (b)functional fragments comprising one or more of these helices. The otherpolypeptide portion of the fusion protein may be contributed by anotherhelical-bundle cytokine or interferon, such as INF-α, or by a non-nativeand/or an unrelated secretory signal peptide that facilitates secretionof the fusion protein.

[0080] The Zcyto21 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides can be produced according to conventional techniques usingcells into which have been introduced an expression vector encoding thepolypeptide. As used herein, “cells into which have been introduced anexpression vector” include both cells that have been directlymanipulated by the introduction of exogenous DNA molecules and progenythereof that contain the introduced DNA. Suitable host cells are thosecell types that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons; Inc., NY, 1987.

[0081] In general, a DNA sequence encoding a Zcyto21 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0082] To direct a Zcyto21 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of Zcyto21, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the Zcyto21 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

[0083] Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter. Expression vectors for use in mammaliancells include pZP-1 and pZP-9, which have been deposited with theAmerican Type Culture Collection, Manassas, Va. USA under accessionnumbers 98669 and 98668, respectively, and derivatives thereof.

[0084] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

[0085] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. In an alternativemethod, adenovirus vector-infected 293 cells can be grown as adherentcells or in suspension culture at relatively high cell density toproduce significant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins can also be effectivelyobtained.

[0086] Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV) according to methods known in the art. Within a preferredmethod, recombinant baculovirus is produced through the use of atransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Bac™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing aZcyto21-encoding sequence is transformed into E. coli host cells, andthe cells are screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.Recombinant virus that expresses Zcyto21 protein is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0087] For protein production, the recombinant virus is used to infecthost cells, typically a cell line derived from the fall armyworm,Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni(e.g., High Five™ cells; Invitrogen, Carlsbad, Calif.). See, forexample, U.S. Pat. No. 5,300,435. Serum-free media are used to grow andmaintain the cells. Suitable media formulations are known in the art andcan be obtained from commercial suppliers. The cells are grown up froman inoculation density of approximately 2-5×10⁵ cells to a density of1-2×10⁶ cells, at which time a recombinant viral stock is added at amultiplicity of infection (MOI) of 0.1 to 10, more typically near 3.Procedures used are generally known in the art.

[0088] Other higher eukaryotic cells can also be used as hosts,including plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.

[0089] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker. commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

[0090] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a Zcyto21polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0091] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

[0092] It is preferred to purify the polypeptides and proteins of thepresent invention to ≧80% purity, more preferably to ≧90% purity, evenmore preferably ≧95% purity, and particularly preferred is apharmaceutically pure state, that is greater than 99.9% pure withrespect to contaminating macromolecules, particularly other proteins andnucleic acids, and free of infectious and pyrogenic agents. Preferably,a purified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

[0093] Expressed recombinant Zcyto21 proteins (including chimericpolypeptides and multimeric proteins) are purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al., ibid.Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

[0094] Zcyto21 polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., SolidPhase, Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford,Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton etal., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,Oxford, 1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

[0095] Using methods known in the art, Zcyto21 proteins can be preparedas monomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

[0096] Target cells for use in Zcyto21 activity assays include, withoutlimitation, vascular cells (especially endothelial cells and smoothmuscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells(including hepatocytes, fenestrated endothelial cells, Kupffer cells,and Ito cells), fibroblasts (including human dermal fibroblasts and lungfibroblasts), fetal lung cells, articular synoviocytes, pericytes,chondrocytes, osteoblasts, and prostate epithelial cells. Endothelialcells and hematopoietic cells are derived from a common ancestral cell,the hemangioblast (Choi et al., Development 125:725-732, 1998).

[0097] Zcyto21 proteins of the present invention are characterized bytheir activity, that is, modulation of the proliferation,differentiation, migration, adhesion, or metabolism of responsive celltypes. Biological activity of Zcyto21 proteins is assayed using in vitroor in vivo assays designed to detect cell proliferation,differentiation, migration or adhesion; or changes in cellularmetabolism (e.g., production of other growth factors or othermacromolecules). Many suitable assays are known in the art, andrepresentative assays are disclosed herein. Assays using cultured cellsare most convenient for screening, such as for determining the effectsof amino acid substitutions, deletions, or insertions. However, in viewof the complexity of developmental processes (e.g., angiogenesis, woundhealing), in vivo assays will generally be employed to confirm andfurther characterize biological activity. Certain in vitro models, suchas the three-dimensional collagen gel matrix model of Pepper et al.(Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficientlycomplex to assay histological effects. Assays can be performed usingexogenously produced proteins, or may be carried out in vivo or in vitrousing cells expressing the polypeptide(s) of interest. Assays can beconducted using Zcyto21 proteins alone or in combination with othergrowth factors, such as members of the VEGF family or hematopoieticcytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representativeassays are disclosed below.

[0098] Activity of Zcyto21 proteins can be measured in vitro usingcultured cells or in vivo by administering molecules of the claimedinvention to an appropriate animal model. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990), incorporation of radiolabelled nucleotides (asdisclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985;Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al.,Analytical Biochem. 179:1-7, 1989). incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use oftetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley etal., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84,1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).Differentiation can be assayed using suitable precursor cells that canbe induced to differentiate into a more mature phenotype. Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

[0099] Zcyto21 activity may also be detected using assays designed tomeasure Zcyto21-induced production of one or more additional growthfactors or other macromolecules. Preferred such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules produced by the liver. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a Zcyto21 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are alsocarried out in the presence of IL-1α or TGF-α to examine the ability ofZcyto21 protein to modify the established responses to these cytokines.

[0100] Monocyte activation assays are carried out (1) to look for theability of Zcyto21 proteins to further stimulate monocyte activation,and (2) to examine the ability of Zcyto21 proteins to modulateattachment-induced or endotoxin-induced monocyte activation (Fuhlbriggeet al., J. Immunol. 138: 3799-3802, 1987). IL-1α and TNFα levelsproduced in response to activation are measured by ELISA (Biosource,Inc. Camarillo, Calif.). Monocyte/macrophage cells, by virtue of CD14(LPS receptor), are exquisitely sensitive to endotoxin, and proteinswith moderate levels of endotoxin-like activity will activate thesecells.

[0101] Hematopoietic activity of Zcyto21 proteins can be assayed onvarious hematopoietic cells in culture. Preferred assays include primarybone marrow colony assays and later stage lineage-restricted colonyassays, which are known in the art (e.g., Holly et al., WIPO PublicationWO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g.,50% methylcellulose containing 15% fetal bovine serum, 10% bovine serumalbumin, and 0.6% PSN antibiotic mix) are incubated in the presence oftest polypeptide, then examined microscopically for colony formation.Known hematopoietic factors are used as controls. Mitogenic activity ofZcyto21 polypeptides on hematopoietic cell lines can be measured asdisclosed above.

[0102] Cell migration is assayed essentially as disclosed by Kähler etal. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939,1997). A protein is considered to be chemotactic if it induces migrationof cells from an area of low protein concentration to an area of highprotein concentration. A typical assay is performed using modifiedBoyden chambers with a polystryrene membrane separating the two chambers(Transwell; Corning Costar Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve. Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997.)

[0103] Cell adhesion activity is assayed essentially as disclosed byLaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,microtiter plates are coated with the test protein, non-specific sitesare blocked with BSA, and cells (such as smooth muscle cells,leukocytes, or endothelial cells) are plated at a density ofapproximately 10⁴-10⁵ cells/well. The wells are incubated at 37° C.(typically for about 60 minutes), then non-adherent cells are removed bygentle washing. Adhered cells are quantitated by conventional methods(e.g., by staining with crystal violet, lysing the cells, anddetermining the optical density of the lysate). Control wells are coatedwith a known adhesive protein, such as fibronectin or vitronectin.

[0104] The activity of Zcyto21 proteins can be measured with asilicon-based biosensor microphysiometer that measures the extracellularacidification rate or proton excretion associated with receptor bindingand subsequent physiologic cellular responses. An exemplary such deviceis the Cytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif.. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including Zcyto21 proteins, their agonists, and antagonists.Preferably, the microphysiometer is used to measure responses of aZcyto21-responsive eukaryotic cell, compared to a control eukaryoticcell that does not respond to Zcyto21 polypeptide. Zcyto21-responsiveeukaryotic cells comprise cells into which a receptor for Zcyto21 hasbeen transfected, thereby creating a cell that is responsive to Zcyto21,as well as cells naturally responsive to Zcyto21. Differences, measuredby a change, for example, an increase or diminution in extracellularacidification, in the response of cells exposed to Zcyto21polypeptide,relative to a control not exposed to Zcyto21, are a direct measurementof Zcyto21-modulated cellular responses. Moreover, suchZcyto21-modulated responses can be assayed under a variety of stimuli.The present invention thus provides methods of identifying agonists andantagonists of Zcyto21 proteins, comprising providing cells responsiveto a Zcyto21 polypeptide, culturing a first portion of the cells in theabsence of a test compound, culturing a second portion of the cells inthe presence of a test compound, and detecting a change, for example, anincrease or diminution, in a cellular response of the second portion ofthe cells as compared to the first portion of the cells. The change incellular response is shown as a measurable change in extracellularacidification rate. Culturing a third portion of the cells in thepresence of a Zcyto21 protein and the absence of a test compoundprovides a positive control for the Zcyto21-responsive cells and acontrol to compare the agonist activity of a test compound with that ofthe Zcyto21 polypeptide. Antagonists of Zcyto21 can be identified byexposing the cells to Zcyto21 protein in the presence and absence of thetest compound, whereby a reduction in Zcyto21-stimulated activity isindicative of antagonist activity in the test compound.

[0105] Expression of Zcyto21 polynucleotides in animals provides modelsfor further study of the biological effects of overproduction orinhibition of protein activity in vivo. Zcyto21-encoding polynucleotidesand antisense polynucleotides can be introduced into test animals, suchas mice, using viral vectors or naked DNA, or transgenic animals can beproduced.

[0106] One in vivo approach for assaying proteins of the presentinvention utilizes viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

[0107] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene is deleted from the viral vector, and the virus willnot replicate unless the E1 gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

[0108] An alternative method of gene delivery comprises removing cellsfrom the body and introducing a vector into the cells as a naked DNAplasmid. The transformed cells are then re-implanted in the body. NakedDNA vectors are introduced into host cells by methods known in the art,including transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, use of agene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol.Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967,1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

[0109] Transgenic mice, engineered to express a Zcyto21 gene, and micethat exhibit a complete absence of Zcyto21 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also begenerated (Lowell et al., Nature 366:740-742, 1993). These mice can beemployed to study the Zcyto21 gene and the protein encoded thereby in anin vivo system. Transgenic mice are particularly useful forinvestigating the role of Zcyto21 proteins in early development in thatthey allow the identification of developmental abnormalities or blocksresulting from the over- or underexpression of a specific factor. Seealso, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science277:48-50, 1997. Preferred promoters for transgenic expression includepromoters from metallothionein and albumin genes.

[0110] A loss of normal inhibitory control of muscle contraction hasbeen associated with damage or perturbation of selectedgamma-aminobutryric acid-secreting neurons. For example, Stiff ManSyndrome exhibit remarkable stiffness of musculature, believed to bemediated through interference of the functioning of theirgamma-aminobutryric acid (GABA) producing neurons. Other relatedneuromuscular disorders include myotonia, metabolic myopathies, Isaac'ssyndrome, dystonia, and tetanic spasms (Valldeoriola, J. Neurol246:423-431, 1999).

[0111] Similarly, direct measurement of Zcyto21 polypeptide, or its lossof expression in a tissue can be determined in a tissue or cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of Zcyto21 in a pre-cancerousor cancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to Zcyto21 expression. For example, appearance or disappearanceof polypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility, oras a liver-specific marker, Zcyto21 gain or loss of expression may serveas a diagnostic for brain and other cancers. Moreover, analogous to theprostate specific antigen (PSA), increased levels of Zcyto21polypeptides, or anti-Zcyto21 antibodies in a patient, relative to anormal control can be indicative of brain and other cancers (See, e.g.,Mulders, TMT, et al., Eur. J. Surgical Oncol. 16:37-41, 1990). StrongZcyto21 expression in tissue not normally found to express Zcyto21 wouldserve as a diagnostic of an abnormality in the cell or tissue type, ofinvasion or metastasis of cancerous liver tissue into non-liver tissue,and could aid a physician in directing further testing or investigation,or aid in directing therapy.

[0112] In addition, Zcyto21 polynucleotide probes, anti-Zcyto21antibodies, and detection the presence of Zcyto21 polypeptides in tissuecan be used to assess whether brain or other tissue found to normallyexpress Zcyto21 is present, for example, after surgery involving theexcision of a diseased or cancerous liver or neuronal tissue. As such,the polynucleotides, polypeptides, and antibodies of the presentinvention can be used as an aid to determine whether all tissue isexcised after surgery, for example, after surgery for brain and othercancers. In such instances, it is especially important to remove allpotentially diseased tissue to maximize recovery from the cancer, and tominimize recurrence. Preferred embodiments include fluorescent,radiolabeled, or calorimetrically labeled anti-Zcyto21 antibodies andZcyto21 polypeptide binding partners, that can be used histologically orin situ.

[0113] Moreover, the activity and effect of Zcyto21 on tumor progressionand metastasis can be measured in vivo. Several syngeneic mouse modelshave been developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6 mouse, that are readily cultured and manipulatedin vitro. Tumors resulting from implantation of either of these celllines are capable of metastasis to the lung in C57BL6 mice. The Lewislung carcinoma model has recently been used in mice to identify aninhibitor of angiogenesis (O'Reilly M S, et al. Cell 79: 315-328,1994).C57BL6/J mice are treated with an experimental agent either throughdaily injection of recombinant protein, agonist or antagonist or aone-time injection of recombinant adenovirus. Three days following thistreatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin.Alternatively, the cells themselves may be infected with recombinantadenovirus, such as one expressing Zcyto21, before implantation so thatthe protein is synthesized at the tumor site or intracellularly, ratherthan stemically. The mice normally develop visible tumors within 5 days.The tumors are allowed to grow for a period of up to 3 weeks, duringwhich time they may reach a size of 1500-1800 mm³ in the control treatedgroup. Tumor size and body weight are carefully monitored throughout theexperiment. At the time of sacrifice, the tumor is removed and weighedalong with the lungs and the liver. The lung weight has been shown tocorrelate well with metastatic tumor burden. As an additional measure,lung surface metastases are counted. The resected tumor, lungs and liverare prepared for histopathological examination, immunohistochemistry,and in situ hybridization, using methods known in the art and describedherein. The influence of the expressed polypeptide in question, e.g.,Zcyto21, on the ability of the tumor to recruit vasculature and undergometastasis can thus be assessed. In addition, aside from usingadenovirus, the implanted cells can be transiently transfected withZcyto21. Use of stable Zcyto21 transfectants as well as use ofinduceable promoters to activate Zcyto21 expression in vivo are known inthe art and can be used in this system to assess Zcyto21 induction ofmetastasis. Moreover, purified Zcyto21 or Zcyto21-conditioned media canbe directly injected in to this mouse model, and hence be used in thissystem. For general reference see, O'Reilly M S, et al. Cell 79:315-328,1994; and Rusciano D, et al. Murine Models of Liver Metastasis. InvasionMetastasis 14:349-361, 1995.

[0114] Antisense methodology can be used to inhibit Zcyto21 genetranscription to examine the effects of such inhibition in vivo.Polynucleotides that are complementary to a segment of aZcyto21-encoding polynucleotide (e.g., a polynucleotide as set forth inSEQ ID NO:1) are designed to bind to Zcyto21-encoding mRNA and toinhibit translation of such mRNA. Such antisense oligonucleotides canalso be used to inhibit expression of Zcyto21 polypeptide-encoding genesin cell culture.

[0115] Most cytokines as well as other proteins produced by activatedlymphocytes play an important biological role in cell differentiation,activation, recruitment and homeostasis of cells throughout the body.Zcyto21 and inhibitors of Zcyto21 activity are expected to have avariety of therapeutic applications. These therapeutic applicationsinclude treatment of diseases which require immune regulation, includingautoimmune diseases such as rheumatoid arthritis, multiple sclerosis,myasthenia gravis, systemic lupus erythematosis, and diabetes. Zcyto21may be important in the regulation of inflammation, and therefore wouldbe useful in treating rheumatoid arthritis, asthma and sepsis. There maybe a role of Zcyto21 in mediating tumorgenesis, whereby a Zcyto21antagonist would be useful in the treatment of cancer. Zcyto21 may beuseful in modulating the immune system, whereby Zcyto21 and Zcyto21antagonists may be used for reducing graft rejection, preventinggraft-vs-host disease, boosting immunity to infectious diseases,treating immunocompromised patients (e.g., HIV⁺ patients), or inimproving vaccines.

[0116] As an interferon-like polypeptide in tissues of brain, islet,prostate, testis, pituitary, placenta, ovarian tumor, lung tumor, rectaltumor and ovarian tumor, as well as a CD3+ cell line, and a virallyinfected prostate epithelial cell line, Zcyto21 is useful to modulateviral infection, tumorigeneses and metastatis in these and othertissues. In such cases, the interferon-like molecule can be released bycell at the site of infection or abnormal cell growth, or as a secretedmolecule, it can migrate to the site from a distant tissue.

[0117] The antiviral properties of Zcyto21 are particularly useful intreating infection with papilloma viruses in vitro and in vivo. Forexample, tumors caused by human papilloma viruses cause benign tumors(i.e., genital warts) as well as malignant tumors such as squamous-cellcarcinomas. Treatment for these conditions commonly is surgery or tissuedestruction. Currently, however, some antiviral/immunomodulatory drugs,including interferon alpha, have been shown effective in reduce tumorsize. See Baker, G.E et al., Dermatol. Clin. Apr. 15: 331-340, 1997.Further, as discussed by Rockley, P. F. et al., (Pharmacol. Ther. 65(2):265-287, 1995), immunologic therapy with interferons can be directedagainst all sites of infection, including clinical, subclinical , andlatent disease. In this example, IFN-alpha, IFN-beta and IFN-gamma havebeen used successfully as monotherapy as well as in combination withother therapies to treat anogenital condyloma acuminatum. Zcyto21 willbe a useful treatment similar to IFN-alpha, IFN-beta and IFN-gamma inthis treatment. Further more, there has been a strong associationbetween certain types of human papilloma virus and cervical cancer.Zcyto21 can be used to detect, monitor and treat cervical cancers.

[0118] As a small, secreted protein in islet cells Zcyto21 can modulatethe growth and differentiation of these cells. Additionally, Zcyto21 maybe useful in treating diabetes and immunological conditions related tothe growth and differentiation of the cells.

[0119] The presence of Zcyto21 in brain and pituitary cells indicatesthat it may also find use in growth and differentiation of these cells.Further, the molecules of the present invention may be responsible fornutritional homeostasis, including behavioral disorders related tofeeding and appetite suppression. Additionally, Zcyto21 molecules mayfind use in treating reproductive disorders in general.

[0120] Zcyto21 polypeptides can be administered alone or in combinationwith other vasculogenic or angiogenic agents, including VEGF. When usingZcyto21 in combination with an additional agent, the two compounds canbe administered simultaneously or sequentially as appropriate for thespecific condition being treated.

[0121] Zcyto21 will be useful in treating tumorgenesis, and thereforewould be useful in the treatment of cancer. A Zcyto21 inhibition ofanti-IgM stimulated normal B-cells and a similar effect is observed inB-cell tumor lines suggest that there may be therapeutic benefit intreating patients with the Zcyto21 in order to induce the B cell tumorcells into a less proliferative state. The ligand could be administeredin combination with other agents already in use including bothconventional chemotherapeutic agents as well as immune modulators suchas interferon alpha. Alpha/beta interferons have been shown to beeffective in treating some leukemias and animal disease models, and thegrowth inhibitory effects of interferon-alpha and Zcyto21 may beadditive for B-cell tumor-derived cell lines.

[0122] The present invention provides a method of reducing proliferationof a neoplastic B or T cells comprising administering to a mammal with aB or T cell neoplasm an amount of a composition of Zcyto21 sufficient toreduce proliferation of the neoplastic B or T cells. Zcyto21 stimulationof lytic NK cells from marrow progenitors and the proliferation ofT-cells following activation of the antigen receptors would enhancetreatment for patients receiving allogenic marrow transplants, andtherefore, Zcyto21 will enhance the generation of anti-tumor responses,with or without the infusion of donor lymphocytes.

[0123] In another aspect, the present invention provides a method ofreducing proliferation of a neoplastic B or T cells comprisingadministering to a mammal with a B or T cell neoplasm an amount of acomposition of Zcyto21 antagonist sufficient to reducing proliferationof the neoplastic B or T cells. Futhermore, the Zcyto21 antagonist canbe a ligand/toxin fusion protein.

[0124] A Zcyto21-saporin fusion toxin may be employed against a similarset of leukemias and lymphomas, extending the range of leukemias thatcan be treated with Zcyto21. Fusion toxin mediated activation of theZcyto21 receptor provides two independent means to inhibit the growth ofthe target cells, the first being identical to the effects seen by theligand alone, and the second due to delivery of the toxin throughreceptor internalization.

[0125] Based on the teachings herein, the interferon-like Zcyto21molecules of the present invention will be useful to detect, monitor ortreat such diverse conditions as hairy cell leukemia, renal cellcarcinoma, basal cell carcinoma, malignant melanoma, AIDS-relatedKaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia,non-Hodgkin's lymphoma, laryngeal papillomatosis, mycosis fungoides,condyloma acuminata, papillomavirus-induced epidermodysplasiverruciformis, chronic hepatitis B, hepatitis C, chronic hepatitis D,and chronic non-A, non-B/C hepatitis. The U.S. Food and DrugAdministration has approved the use of interferon-β to treat multiplesclerosis, a chronic disease of the nervous system. Interferon-γ is usedto treat chronic granulomatous diseases, in which the interferonenhances the patient's immune response to destroy infectious bacterial,fungal, and protozoal pathogens. Clinical studies also indicate thatinterferon-γ may be useful in the treatment of AIDS, leishmaniasis, andlepromatous leprosy

[0126] For pharmaceutical use, Zcyto21 proteins are formulated fortopical or parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. In general, pharmaceuticalformulations will include a Zcyto21 polypeptide in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water, or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Zcyto21 will preferably be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. Dosing is daily or intermittentlyover the period of treatment. Intravenous administration will be bybolus injection or infusion over a typical period of one to severalhours. Sustained release formulations can also be employed. In general,a therapeutically effective amount of Zcyto21 is an amount sufficient toproduce a clinically significant change in the treated condition, suchas a clinically significant change in hematopoietic or immune function,a significant reduction in morbidity, or a significantly increasedhistological score.

[0127] Zcyto21 proteins, agonists, and antagonists are useful formodulating the expansion, proliferation, activation, differentiation,migration, or metabolism of responsive cell types, which include bothprimary cells and cultured cell lines. Of particular interest in thisregard are hematopoietic cells, mesenchymal cells (including stem cellsand mature myeloid and lymphoid cells), endothelial cells, epithelialcells, smooth muscle cells, fibroblasts, hepatocytes, neural cells andembryonic stem cells. Zcyto21 polypeptides are added to tissue culturemedia for these cell types at a concentration of about 10 pg/ml to about100 ng/ml. Those skilled in the art will recognize that Zcyto21 proteinscan be advantageously combined with other growth factors in culturemedia.

[0128] Within the laboratory research field, Zcyto21 proteins can alsobe used as molecular weight standards or as reagents in assays fordetermining circulating levels of the protein, such as in the diagnosisof disorders characterized by over- or under-production of Zcyto21protein or in the analysis of cell phenotype.

[0129] Zcyto21 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of Zcyto21 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of Zcyto21 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofZcyto21-dependent cellular responses. For example, Zcyto21-responsivecell lines can be transfected with a reporter gene construct that isresponsive to a Zcyto21-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a Zcyto21-activated serum response element (SRE) operablylinked to a gene encoding an assayable protein, such as luciferase.Candidate compounds, solutions, mixtures or extracts are tested for theability to inhibit the activity of Zcyto21 on the target cells asevidenced by a decrease in Zcyto21 stimulation of reporter geneexpression. Assays of this type will detect compounds that directlyblock Zcyto21 binding to cell-surface receptors, as well as compoundsthat block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of Zcyto21 binding to receptor usingZcyto21 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled Zcyto21 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

[0130] As used herein, the term “antibodies” includes polyclonalantibodies, monoclonal antibodies, antigen-binding fragments thereofsuch as F(ab′)₂ and Fab fragments, single chain antibodies, and thelike, including genetically engineered antibodies. Non-human antibodiesmay be humanized by grafting non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. One skilledin the art can generate humanized antibodies with specific and differentconstant domains (i.e., different Ig subclasses) to facilitate orinhibit various immune functions associated with particular antibodyconstant domains. Antibodies are defined to be specifically binding ifthey bind to a Zcyto21 polypeptide or protein with an affinity at least10-fold greater than the binding affinity to control (non-Zcyto21)polypeptide or protein. The affinity of a monoclonal antibody can bereadily determined by one of ordinary skill in the art (see, forexample, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

[0131] Methods for preparing polyclonal and monoclonal antibodies arewell known in the art (see for example, Hurrell, J. G. R., Ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla., 1982, which is incorporated herein byreference). Of particular interest are generating antibodies tohydrophilic antigenic sites which include, for example, residues 155(Glu) to 160 (Glu); residues 51 (Lys) to 56 (Ala); residues 50 (Phe) to55 (Asp); residues 140 (Pro) to 145 (Arg); and residues 154 (Gin) to 159(Lys); as shown in SEQ ID NO: 2. As would be evident to one of ordinaryskill in the art, polyclonal antibodies can be generated from a varietyof warm-blooded animals such as horses, cows, goats, sheep, dogs,chickens, rabbits, mice, and rats. The immunogenicity of a Zcyto21polypeptide may be increased through the use of an adjuvant such as alum(aluminum hydroxide) or Freund's complete or incomplete adjuvant.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of a Zcyto21 polypeptide or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

[0132] Alternative techniques for generating or selecting antibodiesinclude in vitro exposure of lymphocytes to Zcyto21 polypeptides, andselection of antibody display libraries in phage or similar vectors(e.g., through the use of immobilized or labeled Zcyto21 polypeptide).Human antibodies can be produced in transgenic, non-human animals thathave been engineered to contain human immunoglobulin genes as disclosedin WIPO Publication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

[0133] A variety of assays to those skilled in the art can be utilizedto detect antibodies which specifically bind to Zcyto21 polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, Westernblot assays, inhibition or competition assays, and sandwich assays.

[0134] Antibodies to Zcyto21 may be used for affinity purification ofthe protein, within diagnostic assays for determining circulating levelsof the protein; for detecting or quantitating soluble Zcyto21polypeptide as a marker of underlying pathology or disease; forimmunolocalization within whole animals or tissue sections, includingimmunodiagnostic applications; for immunohistochemistry; and asantagonists to block protein activity in vitro and in vivo. Antibodiesto Zcyto21 may also be used for tagging cells that express Zcyto21; foraffinity purification of Zcyto21 polypeptides and proteins; inanalytical methods employing FACS; for screening expression libraries;and for generating anti-idiotypic antibodies. Antibodies can be linkedto other compounds, including therapeutic and diagnostic agents, usingknown methods to provide for targeting of those compounds to cellsexpressing receptors for Zcyto21. For certain applications, including invitro and in vivo diagnostic uses, it is advantageous to employ labeledantibodies. Suitable direct tags or labels include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like; indirect tagsor labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies of thepresent invention may also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications(e.g., inhibition of cellproliferation). See, in general, Ramakrishnan et al., Cancer Res.56:1324-1330, 1996.

[0135] Polypeptides and proteins of the present invention can be used toidentify and isolate receptors. Zcyto21 receptors may be involved ingrowth regulation in the liver, blood vessel formation, and otherdevelopmental processes. For example, Zcyto21 proteins and polypeptidescan be immobilized on a column, and membrane preparations run over thecolumn (as generally disclosed in Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp. 195-202). Proteins and polypeptides can also be radiolabeled(Methods Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) orphotoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514,1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and usedto tag specific cell-surface proteins. In a similar manner, radiolabeledZcyto21 proteins and polypeptides can be used to clone the cognatereceptor in binding assays using cells transfected with an expressioncDNA library.

[0136] Zcyto21 polypeptides can also be used to teach analytical skillssuch as mass spectrometry, circular dichroism, to determineconformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing the Zcyto21 canbe given to the student to analyze. Since the amino acid sequence wouldbe known by the instructor, the protein can be given to the student as atest to determine the skills or develop the skills of the student, theinstructor would then know whether or not the student has correctlyanalyzed the polypeptide. Since every polypeptide is unique, theeducational utility of Zcyto21 would be unique unto itself.

[0137] The antibodies which bind specifically to Zcyto21 can be used asa teaching aid to instruct students how to prepare affinitychromatography columns to purify Zcyto21, cloning and sequencing thepolynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. The Zcyto21 gene,polypeptide, or antibody would then be packaged by reagent companies andsold to educational institutions so that the students gain skill in artof molecular biology. Because each gene and protein is unique, each geneand protein creates unique challenges and learning experiences forstudents in a lab practicum. Such educational kits containing theZcyto21 gene, polypeptide, or antibody are considered within the scopeof the present invention. The present invention, thus generallydescribed, will be understood more readily by reference to the followingexamples, which is provided by way of illustration and is not intendedto be limiting of the present invention.

EXAMPLES Example 1

[0138] An expression plasmid containing all or part of a polynucleotideencoding Zcyto21 is constructed via homologous recombination. A fragmentof Zcyto21 cDNA is isolated by PCR using the polynucleotide sequence ofSEQ ID NO: 1 with flanking regions at the 5′ and 3′ ends correspondingto the vector sequences flanking the Zcyto21 insertion point. Theprimers for PCR each include from 5′ to 3′ end: 40 bp of flankingsequence from the vector and 17 bp corresponding to the amino andcarboxyl termini from the open reading frame of Zcyto21.

[0139] Ten μl of the 100 μl PCR reaction mixture is run on a 0.8%low-melting-temperature agarose (SeaPlaque GTG®; FMC BioProducts,Rockland, Me.) gel with 1×TBE buffer for analysis. The remaining 90 μlof the reaction misture is precipitated with the addition of 5 μl 1 MNaCl and 250 μl of absolute ethanol. The plasmid pZMP6, which has beencut with SmaI, is used for recombination with the PCR fragment. PlamidpZMP6 is a mammalian expression vector containing an expression cassettehaving the cytomegalovirus immediate early promoter, multiplerestriction sites for insertion of coding sequences, a stop codon, and ahuman growth hormone terminator; an E. coli origin of replication; amammalian selectable marker expression unit comprising an SV40 promoter,enhancer and origin of replication, a DHFR gene, and the SV40terminator; and URA3 and CEN-ARS sequences required for selection andreplication in S. cerevisiae. It was constructed from pZP9 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 98668) with the yeastgenetic elements taken from pRS316 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Accession No. 77145), an internal ribosome entry site(IRES) element from poliovirus, and the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain.

[0140] One hundred microliters of competent yeast (S. cerevisiae) cellsare independently combined with 10 μl of the various DNA mixtures fromabove and transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply (BioRad Laboratories,Hercules, Calif.) settings of 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated intwo 300-μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura⁺ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid-washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA is precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet is resuspended in10 μl H₂O.

[0141] Transformation of electrocompetent E. coli host cells (ElectromaxDH10B™ cells; obtained from Life Technologies, Inc., Gaithersburg, Md.)is done with 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

[0142] Individual clones harboring the correct expression construct forZcyto21 are identified by restriction digest to verify the presence ofthe Zcyto21 insert and to confirm that the various DNA sequences havebeen joined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated pZMP6/Zcyto21.

Example 2

[0143] CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet.12:555-666, 1986) are plated in 10-cm tissue culture dishes and allowedto grow to approximately 50% to 70% confluency overnight at 37° C., 5%CO₂, in Ham's F12/FBS media (Ham's F12 medium (Life Technologies), 5%fetal bovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRHBiosciences, Lenexa, Kans.), 1% sodium pyruvate (Life Technologies)).The cells are then transfected with the plasmid Zcyto21/pZMP6 byliposome-mediated transfection using a 3:1 (w/w) liposome formulation ofthe polycationic lipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filetered water (Lipofectamine™ Tm Reagent, Life Technologies),in serum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zcyto21/pZMP6 is diluted into 15-ml tubes to a total final volume of 640μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SFmedium. The resulting mixture is added to the DNA mixture and allowed toincubate approximately 30 minutes at room temperature. Five ml of SFmedia is added to the DNA:Lipofectamine™ mixture. The cells are rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight, and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 postransfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). TheCD8-positive cells are separated using commercially available columns(mini-MACS columns; Miltenyi Biotec) according to the manufacturer'sdirections and put into DMEM/Ham's F12/5% FBS without nucleosides butwith 50 nM methotrexate (selection medium).

[0144] Cells are plated for subcloning at a density of 0.5, 1 and 5cells per well in 96-well dishes in selection medium and allowed to growout for approximately two weeks. The wells are checked for evaporationof medium and brought back to 200 μl per well as necessary during thisprocess. When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is then washed three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filteris developed with chemiluminescence reagents (ECL™ direct labelling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham Corp.) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to 6-well dishes in selection medium forscaleup and analysis by Western blot.

Example 3

[0145] Full-length Zcyto21 protein is produced in BHK cells transfectedwith pZMP6/Zcyto21 (Example 1). BHK 570 cells (ATCC CRL-10314) areplated in 10-cm tissue culture dishes and allowed to grow toapproximately 50 to 70% confluence overnight at 37° C., 5% CO₂, inDMEM/FBS media (DMEM, Gibco/BRL High Glucose; Life Technologies), 5%fetal bovine serum (Hyclone, Logan, Utah), 1 mM L-glutamine (JRHBiosciences, Lenexa, Kans.), 1 mM sodium pyruvate (Life Technologies).The cells are then transfected with pZMP6/Zcyto21 by liposome-mediatedtransfection (using Lipofectamine™; Life Technologies), in serum free(SF) media (DMEM supplemented with 10 mg/ml transferrin, 5 mg/mlinsulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). Theplasmid is diluted into 15-ml tubes to a total final volume of 640 μlwith SF media. 35 μl of the lipid mixture is mixed with 605 μl of SFmedium, and the resulting mixture is allowed to incubate approximately30 minutes at room temperature. Five milliliters of SF media is thenadded to the DNA:lipid mixture. The cells are rinsed once with 5 ml ofSF media, aspirated, and the DNA:lipid mixture is added. The cells areincubated at 37° C. for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSNmedia is added to each plate. The plates are incubated at 37° C.overnight, and the DNA:lipid mixture is replaced with fresh 5% FBS/DMEMmedia the next day. On day 5 post-transfection, the cells are split intoT-162 flasks in selection medium (DMEM+5% FBS, 1% L-Gln, 1% NaPyr, 1 μMmethotrexate). Approximately 10 days post-transfection, two 150-mmculture dishes of methotrexate-resistant colonies from each transfectionare trypsinized, and the cells are pooled and plated into a T-162 flaskand transferred to large-scale culture.

Example 4

[0146] For construction of adenovirus vectors, the protein coding regionof human Zcyto21 is amplified by PCR using primers that add PmeI andAscI restriction sites at the 5′ and 3′ termini respectively.Amplification is performed with a full-length Zcyto21 cDNA template in aPCR reaction as follows: one cycle at 95° C. for 5 minutes; followed by15 cycles at 95° C. for 1 min., 61° C. for 1 min., and 72° C. for 1.5min.; followed by 72° C. for 7 min.; followed by a 4° C. soak. The PCRreaction product is loaded onto a 1.2% low-melting-temperature agarosegel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The Zcyto21 PCRproduct is excised from the gel and purified using a commerciallyavailable kit comprising a silica gel mambrane spin column (QIAquick®PCR Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kitinstructions. The PCR product is then digested with PmeI and AscI,phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 mlTE (Tris/EDTA pH 8). The Zcyto21 fragment is then ligated into thePmeI-AscI sites of the transgenic vector pTG12-8 and transformed into E.coli DH10B™ competent cells by electroporation. Vector pTG12-8 wasderived from p2999B4 (Palmiter et al., Mol. Cell Biol. 13:5266-5275,1993) by insertion of a rat insulin II intron (ca. 200 bp) andpolylinker (Fse I/Pme I/Asc I) into the Nru I site. The vector comprisesa mouse metallothionein (MT-1) promoter (ca. 750 bp) and human growthhormone (hGH) untranslated region and polyadenylation signal (ca. 650bp) flanked by 10 kb of MT-1 5′ flanking sequence and 7 kb of MT-1 3′flanking sequence. The cDNA is inserted between the insulin II and hGHsequences. Clones containing Zcyto21 are identified by plasmid DNAminiprep followed by digestion with PmeI and AscI. A positive clone issequenced to insure that there were no deletions or other anomalies inthe construct.

[0147] DNA is prepared using a commercially available kit (Maxi Kit,Qiagen, Inc.), and the Zcyto21 cDNA is released from the pTG12-8 vectorusing PmeI and AscI enzymes. The cDNA is isolated on a 1% low meltingtemperature agarose gel and excised from the gel. The gel slice ismelted at 70 μC, and the DNA is extracted twice with an equal volume ofTris-buffered phenol, precipitated with EtOH, and resuspended in 10 μlH₂O.

[0148] The Zcyto21 cDNA is cloned into the EcoRV-AscI sites of amodified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA95:2509-2514, 1998). This construct contains the green fluorescentprotein (GFP) marker gene. The CMV promoter driving GFP expression isreplaced with the SV40 promoter, and the SV40 polyadenylation signal isreplaced with the human growth hormone polyadenylation signal. Inaddition, the native polylinker is replaced: with FseI, EcoRV, and AscIsites. This modified form of pAdTrack-CMV is named pZyTrack. Ligation isperformed using a commercially available DNA ligation and screening kit(Fast-Link® kit; Epicentre Technologies, Madison, Wis.). Clonescontaining Zcyto21 are identified by digestion of mini prep DNA withFseI and AscI. In order to linearize the plasmid, approximately 5 μg ofthe resulting pZyTrack Zcyto21 plasmid is digested with PmeI.Approximately 1 μg of the linearized plasmid is cotransformed with 200ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183 cells(He et al., ibid.). The co-transformation is done using a Bio-Rad GenePulser at 2.5 kV, 200 ohms and 25 μFa. The entire co-transformationmixture is plated on 4 LB plates containing 25 μg/ml kanamycin. Thesmallest colonies are picked and expanded in LB/kanamycin, andrecombinant adenovirus DNA is identified by standard DNA miniprepprocedures. The recombinant adenovirus miniprep DNA is transformed intoE. coli DH10B™ competent cells, and DNA is prepared using a Maxi Kit(Qiagen, Inc.) aaccording to kit instructions.

[0149] Approximately 5 μg of recombinant adenoviral DNA is digested withPacI enzyme (New England Biolabs) for 3 hours at 37° C. in a reactionvolume of 100 μl containing 20-30U of PacI. The digested DNA isextracted twice with an equal volume of phenol/chloroform andprecipitated with ethanol. The DNA pellet is resuspended in 10 μldistilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies,Inc. Montreal, Qc. Canada), inoculated the day before and grown to60-70% confluence, is transfected with the PacI digested DNA. ThePacI-digested DNA is diluted up to a total volume of 50 μl with sterileHBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP)(Boehringer Mannheim, Indianapolis, Ind.) is diluted to a total volumeof 100 μl with HBS. The DNA is added to the DOTAP, mixed gently bypipeting up and down, and left at room temperature for 15 minutes. Themedia is removed from the 293A cells and washed with 5 ml serum-freeminimum essential medium (MEM) alpha containing 1 mM sodium pyruvate,0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (reagentsobtained from Life Technologies, Gaithersburg, Md.). 5 ml of serum-freeMEM is added to the 293A cells and held at 37° C. The DNA/lipid mixtureis added drop-wise to the T25 flask of 293A cells, mixed gently, andincubated at 37° C. for 4 hours. After 4 hours the media containing theDNA/lipid mixture is aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells are monitoredfor GFP expression and formation of foci (viral plaques).

[0150] Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells express the GFP protein and start to form foci(viral “plaques”). The crude viral lysate is collected using a cellscraper to collect all of the 293A cells. The lysate is transferred to a50-ml conical tube. To release most of the virus particles from thecells, three freeze/thaw cycles are done in a dry ice/ethanol bath and a37° C. waterbath.

[0151] The crude lysate is amplified (Primary (1°) amplification) toobtain a working “stock” of Zcyto21 rAdV lysate. Ten 10 cm plates ofnearly confluent (80-90%) 293A cells are set up 20 hours previously, 200ml of crude rAdV lysate is added to each 10-cm plate, and the cells aremonitored for 48 to 72 hours for CPE (cytopathic effect) under the whitelight microscope and expression of GFP under the fluorescent microscope.When all of the 293A cells show CPE, this stock lysate is collected andfreeze/thaw cycles performed as described above.

[0152] A secondary (2°) amplification of Zcyto21 rAdV is then performed.Twenty 15-cm tissue culture dishes of 293A cells are prepared so thatthe cells are 80-90% confluent. All but 20 ml of 5% MEM media isremoved, and each dish is inoculated with 300-500 ml of the 1° amplifiedrAdv lysate. After 48 hours the 293A cells are lysed from virusproduction, the lysate is collected into 250-ml polypropylene centrifugebottles, and the rAdV is purified.

[0153] NP-40 detergent is added to a final concentration of 0.5% to thebottles of crude lysate in order to lyse all cells. Bottles are placedon a rotating platform for 10 minutes agitating as fast as possiblewithout the bottles falling over. The debris is pelleted bycentrifugation at 20,000×G for 15 minutes. The supernatant istransferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volumeof 20% PEG8000/2.5 M NaCl solution is added. The bottles are shakenovernight on ice. The bottles are centrifuged at 20,000×G for 15minutes, and the supernatant is discarded into a bleach solution. Usinga sterile cell scraper, the white, virus/PEG precipitate from 2 bottlesis resuspended in 2.5 ml PBS. The resulting virus solution is placed in2-ml microcentrifuge tubes and centrifuged at 14,000×G in themicrocentrifuge for 10 minutes to remove any additional cell debris. Thesupernatant from the 2-ml microcentrifuge tubes is transferred into a15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/mlwith CsCl. The solution is transferred to 3.2-ml, polycarbonate,thick-walled centrifuge tubes and spun at 348,000×G for 3-4 hours at 25μC. The virus forms a white band. Using wide-bore pipette tips, thevirus band is collected.

[0154] A commercially available ion-exchange columns (e.g., PD-10columns prepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway,N.J.) is used to desalt the virus preparation. The column isequilibrated with 20 ml of PBS. The virus is loaded and allowed to runinto the column 5 ml of PBS is added to the column, and fractions of8-10 drops are collected. The optical densities of 1:50 dilutions ofeach fraction are determined at 260 nm on a spectrophotometer. Peakfractions are pooled, and the optical density (OD) of a 1:25 dilution isdetermined. OD is converted to virus concentration using the formula:(OD at 260 nm)(25)(1.1×10¹²)=virions/ml.

[0155] To store the virus, glycerol is added to the purified virus to afinal concentration of 15%, mixed gently but effectively, and stored inaliquots at −80 μC.

[0156] A protocol developed by Quantum Biotechnologies, Inc. (Montreal,Canada) is followed to measure recombinant virus infectivity. Briefly,two 96-well tissue culture plates are seeded with 1×10⁴ 293A cells perwell in MEM containing 2% fetal bovine serum for each recombinant virusto be assayed. After 24 hours 10-fold dilutions of each virus from1×10⁻² to 1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100μl of each dilution is placed in each of 20 wells. After 5 days at 37°C., wells are read either positive or negative for CPE, and a value for“Plaque Forming Units/ml” (PFU) is calculated.

Example 5

[0157] Trangenic animals expressing Zcyto21 genes are producing usingadult, fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms,Germantown, N.Y.)), vasectomized males (duds) (CD1, 2-8 months, (TaconicFarms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks,(Taconic Farms)) and adult fertile females (recipients) (CD1, 2-4months, (Taconic Farms)).

[0158] The donors are acclimated for 1 week and then injected withapproximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma,St. Louis, Mo.) I.P., and 46-47 hours later, 8 lU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

[0159] Fertilized eggs are collected under a surgical scope (Leica MZ12Stereo Microscope, Leica, Wetzlar, Del.). The oviducts are collected andeggs are released into urinanalysis slides containing hyaluronidase(Sigma). Eggs are washed once in hyaluronidase, and twice in Whitten'sW640 medium (Table 4) that has been incubated with 5% CO₂, 5% O₂, and90% N₂ at 37° C. The eggs are then stored in a 37° C./5% CO₂ incubatoruntil microinjection.

[0160] 10-20 micrograms of plasmid DNA containing a cDNA of the Zcyto21gene is linearized, gel-purified, and resuspended in 10 mM Tris pH 7.4.0.25 mM EDTA pH 8.0, at a final concentration of 5-10 nanograms permicroliter for microinjection.

[0161] Plasmid DNA is microinjected into harvested eggs contained in adrop of W640 medium overlaid by warm, CO₂-equilibrated mineral oil. TheDNA is drawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

[0162] Picoliters of DNA are injected into the pronuclei, and theinjection needle withdrawn without coming into contact with thenucleoli. The procedure is repeated until all the eggs are injected.Successfully microinjected eggs are transferred into an organtissue-culture dish with pregassed W640 medium for storage overnight ina 37° C./5% CO₂ incubator.

[0163] The following day, 12-17 healthy 2-cell embryos from the previousday's injection are transferred into the recipient. The swollen ampullais located and holding the oviduct between the ampulla and the bursa, anick in the oviduct is made with a 28 g needle close to the bursa,making sure not to tear the ampulla or the bursa. The embryos areimplanted through this nick, and by holding onto the peritoneal wall,the reproductive organs are guided back into the abdominal cavity.

[0164] The recipients are returned to cages in pairs, and allowed 19-21days gestation. After birth, 19-21 days postpartum is allowed beforeweaning. The weanlings are sexed and placed into separate sex cages, anda 0.5 cm biopsy (used for genotyping) is snipped off the tail with cleanscissors.

[0165] Genomic DNA is prepared from the tail snips using a Qiagen Dneasykit following the manufacturer's instructions. Genomic DNA is analyzedby PCR using primers designed to the human growth hormone (hGH) 3′ UTRportion of the transgenic vector. A region unique to the human sequencewas identified from an alignment of the human and mouse growth hormone3′ UTR DNA sequences, ensuring that the PCR reaction does not amplifythe mouse sequence. Primers which amplify a 368 base pair fragment ofhGH and primers which hybridize to vector sequences and amplify the cDNAinsert, are often used along with the hGH primers. In these experiments,DNA from animals positive for the transgene will generate two bands, a368 base pair band corresponding to the hGH 3′ UTR fragment and a bandof variable size corresponding to the cDNA insert.

[0166] Once animals are confirmed to be transgenic (TG), they may beback-crossed into an inbred strain by placing a TG female with awild-type male, or a TG male with one or two wild-type female(s). Aspups are born and weaned, the sexes are separated, and their tailssnipped for genotyping.

[0167] Analysis of the mRNA expression level of each transgene is doneusing an RNA solution hybridization assay or real-time PCR on an ABIPrism 7700 (PE Applied Biosystems, Inc., Foster City, Calif.) followingmanufacturer's instructions. TABLE 5 WHITTEN'S 640 MEDIA mgs/200 mmgs/500/ml NaCl 1280 3200 KCl 72 180 KH₂PO₄ 32 80 MgSO₄.7H₂O 60 150Glucose 200 500 Ca²⁺ Lactate 106 265 K Penn 15 37.5 Streptomycin SO₄ 1025 NaHCO₃ 380 950 Na Pyruvate 5 12.5 H₂O 200 500 EDTA 100 μl 250 μl 5%Phenol Red 200 μl 500 μl BSA 600 1500

Example 6

[0168] 1. Stimulation of Expression from an Interferon-ResponsivePromoter

[0169] In one series of experiments, conditioned medium (CM) containingZcyto21 protein is generated by infecting 293A cells with recombinantadenovirus containing the cDNA for Zcyto21 (AdZy-Zcyto21) at amultiplicity of infection of 400 particles per cell. CM is harvested attime points between 40 hours post infection and stored at −20° C. CM isalso generated from an infection with a recombinant adenovirus lacking acDNA (AdZy-parental). Prior to use, a portion of the CM is concentrated14 fold in a Millipore Ultrafree-15 (5,000 nominal molecular weightlimit) centrifugal filter, and then, filtered through a MilliporeUltrafree-15 (100,000 nominal molecular weight limit) centrifugal filterto reduce the amount of viral particles present in the media, andfinally filtered through a Millipore 0.2 μm syringe filter to sterilizethe CM. Concentrated CM samples are diluted 1:2 in binding buffer andincubated with cells from a murine cell line for 5 hours at 37° C.

[0170] 2. Anti-Viral Activity of Zcyto21

[0171] Another series of experiments examines the anti-viral activity ofZcyto21. In these studies, the anti-viral assay is performed by platingL929 cells (ATCC No. CCL-1) in growth media RPMI medium 1640 containing10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in96-well format at 50,000 cells per well. Adenovirus CM from 293A cellsinfected with either AdZy-Zcyto21m or AdZy-parental, as described above,are incubated with cells overnight. A positive control in the assay isprovided by murine interferon-α serially diluted 1:10, starting at 100ng/ml. L929 cells with growth media alone provided the negative control.Treated cells are incubated for 24 hours. The media are discarded, freshmedium are added, and encephalomyocarditis virus (ATCC No. vr129b) isintroduced at a multiplicity of infection of 0.1 (i.e., one virusparticle for every ten L929 cells). The cells are incubated in thepresence of the virus for 24 hours, and then, the wells are scored forpercent cytopathic effect (CPE

[0172] 3. Antiproliferation Assay Using a BAF3 Cell Line

[0173] BaF3 is used to determine if Zcyto21 has anti-proliferativeproperties. Baby hamster kidney (BHK) cells are stably transfected withan expression vector containing the CMV promoter plus intron A upstreamof the Zcyto21 cDNA or an unrelated cDNA, called Zα30, using BRLlipofectamine. Stably transfected cells are seeded in a cell factory inserum free media and allowed to grow for three days before conditionedmedia is harvested and concentrated in a 5K filter to 10×. Concentratedconditioned medium samples are stored at 4° C.

[0174] The following assay is used to test for anti-proliferation ofBaF3. In a 96 well plate, eight 1:2 serial dilutions are made of growthmedia alone (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 2mM L-glutamine), or murine IL-3 (starting at 50 pg/ml in growth medium)with final volume of 100 μl. Fifty microliters of the following areadded to both growth media alone or mIL-3 diluted lanes: humaninterferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium),human interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growthmedium), murine interferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted ingrowth medium), murine interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/mldiluted in growth medium), Zcyto21 (at 2.5×, 0.5×, or 0.1×), and murineZα30 (at 2.5×, 0.5×, or 0.1×).

[0175] The BaF3cell line is washed three times in growth medium, pelletsare resuspended in growth medium, cells are counted and diluted ingrowth medium to 5,000 cells/50 μl. Fifty microliters of diluted cellsare then added to each dilution of samples. Assay plates are incubatedin a 37° C. incubator for three to four days. Twenty microliters ofAlomar blue are then added to each well and the plate are incubatedovernight at 37° C. The plates are read on the fluorescent plate readerat excitation wavelength of 544 and emission wavelength 590.

Example 7

[0176] Tissue Distribution of Human Zcyto21 in Tissue Panels Using PCR

[0177] A panel of cDNA samples from human tissues was screened forZcyto21 expression using PCR. The panel was made in house and contained77 marathon cDNA and cDNA samples from various norman and canceroushuman tissues and cell lines as shown in Table 6, below. The cDNAsamples came from in-house libraries or marathon cDNA preparations ofRNA that were prepared in-house, or from a commercial supplier such asClontech (Palo Alto, Calif.) or Invitrogen (Carlsbad, Calif.). Themarathon cDNAs were made using the Marathon cDNA Amplification Kit(Clontech). To assure quality of the panel samples, three tests forquality control (QC) were run: (1) To assess the RNA quality used forthe libraries, the in-house cDNAs were tested for average insert size byPCR with vector oligos that were specific for the vector sequences foran individual cDNA library; (2) Standardization of the concentration ofthe cDNA in panel samples was achieved using standard PCR methods toamplify full length alpha tubulin or G3PDH cDNA; and (3) a sample wassent to sequencing to check for possible ribosomal or mitochondrial DNAcontamination. The panel was set up in a 96-well format that included ahuman genomic DNA (Clontech) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The first PCR reactionswere set up using oligos ZC39,270 ( SEQ ID NO:14) and ZC39,272 (SEQ IDNO:15), Advantage 2 DNA Polymerase Mix (Clontech) and Rediload dye(Research Genetics, Inc., Huntsville, Ala.). The amplification wascarried out as follows: 1 cycle at 94° for 1 minute then 35 cycles of94°, 10 seconds; 67°, 45 seconds and ended with a 3 minute finalextension at 72°. The correct DNA fragment size was observed in brain,islet, prostate, testis, pituitary, placenta, ovarian tumor, lung tumor,CD3+ and HPVS. Another PCR reaction was set up using oligos ZC39,270(SEQ ID NO:14) and ZC39,271 ( SEQ ID NO:16), Advantage 2 DNA PolymeraseMix (Clontech) and Rediload dye (Research Genetics). The amplificationwas carried out as follows: 1 cycle at 94°, 1 minute then 35 cycles of94°, 10 seconds; 65°, 30 seconds, 72°, 30 seconds and ended with a 3minute extension at 72°. The correct DNA fragment size was observed inpituitary, rectal tumor and ovarian tumor. TABLE 6 # # samples samplesTissue tested Tissue tested adrenal gland 1 bladder 1 bone marrow 3brain 2 cervix 1 colon 1 fetal brain 3 fetal heart 2 fetal kidney 1fetal liver 2 fetal lung 1 fetal skin 1 heart 2 fetal muscle 1 kidney 2liver 1 lung 1 lymph node 1 mammary gland 1 melanoma 1 ovary 1 pancreas1 pituitary 2 placenta 3 prostate 3 rectum 1 salivary gland 2 skeletalmuscle 1 small intestine 1 spinal cord 2 spleen 1 uterus 1 stomach 1adipocyte library 1 testis 5 islet 1 thymus 1 prostate SMC 1 thyroid 2RPMI 1788 (ATCC # CCL- 1 156) trachea 1 WI38 (ATCC # CCL-75) 1esophageal tumor 1 lung tumor 1 liver tumor 1 ovarian tumor 1 rectaltumor 1 stomach tumor 1 uterine tumor 2 CD3+ library selected 1 PBMC's(stimulated) HaCAT library 1 HPV library(ATCC # 1 CRL-2221) HPVSlibrary(ATCC # 1 MG63 library 1 CRL-2221) - selected K562 (ATCC#CCL- 1243)

[0178] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 16 1 603 DNA Homo sapiens CDS (1)...(603) 1 atg gct gca gct tgg accgtg gtg ctg gtg act ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr ValVal Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 gcc gtg gca ggc cct gtcccc act tcc aag ccc acc aca act ggg aag 96 Ala Val Ala Gly Pro Val ProThr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 ggc tgc cac att ggc agg ttcaaa tct ctg tca cca cag gag cta gcg 144 Gly Cys His Ile Gly Arg Phe LysSer Leu Ser Pro Gln Glu Leu Ala 35 40 45 agc ttc aag aag gcc agg gac gccttg gaa gag tca ctc aag ctg aaa 192 Ser Phe Lys Lys Ala Arg Asp Ala LeuGlu Glu Ser Leu Lys Leu Lys 50 55 60 aac tgg agt tgc agc tct cct gtc ttcccc ggg aat tgg gac ctg agg 240 Asn Trp Ser Cys Ser Ser Pro Val Phe ProGly Asn Trp Asp Leu Arg 65 70 75 80 ctt ctc cag gtg agg gag cgc cct gtggcc ttg gag gct gag ctg gcc 288 Leu Leu Gln Val Arg Glu Arg Pro Val AlaLeu Glu Ala Glu Leu Ala 85 90 95 ctg acg ctg aag gtc ctg gag gcc gct gctggc cca gcc ctg gag gac 336 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala GlyPro Ala Leu Glu Asp 100 105 110 gtc cta gac cag ccc ctt cac acc ctg caccac atc ctc tcc cag ctc 384 Val Leu Asp Gln Pro Leu His Thr Leu His HisIle Leu Ser Gln Leu 115 120 125 cag gcc tgt atc cag cct cag ccc aca gcaggg ccc agg ccc cgg ggc 432 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala GlyPro Arg Pro Arg Gly 130 135 140 cgc ctc cac cac tgg ctg cac cgg ctc caggag gcc ccc aaa aag gag 480 Arg Leu His His Trp Leu His Arg Leu Gln GluAla Pro Lys Lys Glu 145 150 155 160 tcc gct ggc tgc ctg gag gca tct gtcacc ttc aac ctc ttc cgc ctc 528 Ser Ala Gly Cys Leu Glu Ala Ser Val ThrPhe Asn Leu Phe Arg Leu 165 170 175 ctc acg cga gac ctc aaa tat gtg gccgat ggg gac ctg tgt ctg aga 576 Leu Thr Arg Asp Leu Lys Tyr Val Ala AspGly Asp Leu Cys Leu Arg 180 185 190 acg tca acc cac cct gag tcc acc tga603 Thr Ser Thr His Pro Glu Ser Thr * 195 200 2 200 PRT Homo sapiens 2Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 1015 Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 2530 Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 4045 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 5560 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65 7075 80 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala 8590 95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser GlnLeu 115 120 125 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg ProArg Gly 130 135 140 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala ProLys Lys Glu 145 150 155 160 Ser Ala Gly Cys Leu Glu Ala Ser Val Thr PheAsn Leu Phe Arg Leu 165 170 175 Leu Thr Arg Asp Leu Lys Tyr Val Ala AspGly Asp Leu Cys Leu Arg 180 185 190 Thr Ser Thr His Pro Glu Ser Thr 195200 3 600 DNA Artificial Sequence degenerate sequence 3 atggcngcngcntggacngt ngtnytngtn acnytngtny tnggnytngc ngtngcnggn 60 ccngtnccnacnwsnaarcc nacnacnacn ggnaarggnt gycayathgg nmgnttyaar 120 wsnytnwsnccncargaryt ngcnwsntty aaraargcnm gngaygcnyt ngargarwsn 180 ytnaarytnaaraaytggws ntgywsnwsn ccngtnttyc cnggnaaytg ggayytnmgn 240 ytnytncargtnmgngarmg nccngtngcn ytngargcng arytngcnyt nacnytnaar 300 gtnytngargcngcngcngg nccngcnytn gargaygtny tngaycarcc nytncayacn 360 ytncaycayathytnwsnca rytncargcn tgyathcarc cncarccnac ngcnggnccn 420 mgnccnmgnggnmgnytnca ycaytggytn caymgnytnc argargcncc naaraargar 480 wsngcnggntgyytngargc nwsngtnacn ttyaayytnt tymgnytnyt nacnmgngay 540 ytnaartaygtngcngaygg ngayytntgy ytnmgnacnw snacncaycc ngarwsnacn 600 4 603 DNAHomo sapiens CDS (1)...(603) 4 atg gct gca gct tgg acc gtg gtg ctg gtgact ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr Val Val Leu Val ThrLeu Val Leu Gly Leu 1 5 10 15 gcc gtg gca ggc cct gtc ccc act tcc aagccc acc aca act ggg aag 96 Ala Val Ala Gly Pro Val Pro Thr Ser Lys ProThr Thr Thr Gly Lys 20 25 30 ggc tgc cac att ggc agg ttc aaa tct ctg tcacca cag gag cta gcg 144 Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser ProGln Glu Leu Ala 35 40 45 agc ttc aag aag gcc agg gac gcc ttg gaa gag tcactc aag ctg aaa 192 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser LeuLys Leu Lys 50 55 60 aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgggac ctg agg 240 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp AspLeu Arg 65 70 75 80 ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gctgag ctg gcc 288 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala GluLeu Ala 85 90 95 ctg acg ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctggag gac 336 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu GluAsp 100 105 110 gtc cta gac cag ccc ctt cac acc ctg cac cac atc ctc tcccag ctc 384 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser GlnLeu 115 120 125 cag gcc tgt atc cag cct cag ccc aca gca ggg ccc agg ccccgg ggc 432 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro ArgGly 130 135 140 cgc ctc cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaaaag gag 480 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys LysGlu 145 150 155 160 tcc gct ggc tgc ctg gag gca tct gtc acc ttc aac ctcttc cgc ctc 528 Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu PheArg Leu 165 170 175 ctc acg cga gac ctc aaa tat gtg gcc gat ggg aac ctgtgt ctg aga 576 Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu CysLeu Arg 180 185 190 acg tca acc cac cct gag tcc acc tga 603 Thr Ser ThrHis Pro Glu Ser Thr * 195 200 5 200 PRT Homo sapiens 5 Met Ala Ala AlaTrp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 Ala Val AlaGly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly Cys HisIle Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 Ser Phe LysLys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60 Asn Trp SerCys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65 70 75 80 Leu LeuGln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala 85 90 95 Leu ThrLeu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp 100 105 110 ValLeu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 115 120 125Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly 130 135140 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145150 155 160 Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe ArgLeu 165 170 175 Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu CysLeu Arg 180 185 190 Thr Ser Thr His Pro Glu Ser Thr 195 200 6 856 DNAHomo sapiens CDS (98)...(700) 6 aattaccttt tcactttaca cacatcatcttggattgccc attttgcgtg gctaaaaagc 60 agagccatgc cgctggggaa gcagttgcgatttagcc atg gct gca gct tgg acc 115 Met Ala Ala Ala Trp Thr 1 5 gtg gtgctg gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct gtc 163 Val Val LeuVal Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro Val 10 15 20 ccc act tccaag ccc acc aca act ggg aag ggc tgc cac att ggc agg 211 Pro Thr Ser LysPro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg 25 30 35 ttc aaa tct ctgtca cca cag gag cta gcg agc ttc aag aag gcc agg 259 Phe Lys Ser Leu SerPro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg 40 45 50 gac gcc ttg gaa gagtca ctc aag ctg aaa aac tgg agt tgc agc tct 307 Asp Ala Leu Glu Glu SerLeu Lys Leu Lys Asn Trp Ser Cys Ser Ser 55 60 65 70 cct gtc ttc ccc gggaat tgg gac ctg agg ctt ctc cag gtg agg gag 355 Pro Val Phe Pro Gly AsnTrp Asp Leu Arg Leu Leu Gln Val Arg Glu 75 80 85 cgc cct gtg gcc ttg gaggct gag ctg gcc ctg acg ctg aag gtc ctg 403 Arg Pro Val Ala Leu Glu AlaGlu Leu Ala Leu Thr Leu Lys Val Leu 90 95 100 gag gcc gct gct ggc ccagcc ctg gag gac gtc cta gac cag ccc ctt 451 Glu Ala Ala Ala Gly Pro AlaLeu Glu Asp Val Leu Asp Gln Pro Leu 105 110 115 cac acc ctg cac cac atcctc tcc cag ctc cag gcc tgt atc cag cct 499 His Thr Leu His His Ile LeuSer Gln Leu Gln Ala Cys Ile Gln Pro 120 125 130 cag ccc aca gca ggg cccagg ccc cgg ggc cgc ctc cac cac tgg ctg 547 Gln Pro Thr Ala Gly Pro ArgPro Arg Gly Arg Leu His His Trp Leu 135 140 145 150 cac cgg ctc cag gaggcc ccc aaa aag gag tcc gct ggc tgc ctg gag 595 His Arg Leu Gln Glu AlaPro Lys Lys Glu Ser Ala Gly Cys Leu Glu 155 160 165 gca tct gtc acc ttcaac ctc ttc cgc ctc ctc acg cga gac ctc aaa 643 Ala Ser Val Thr Phe AsnLeu Phe Arg Leu Leu Thr Arg Asp Leu Lys 170 175 180 tat gtg gcc gat gggaac ctg tgt ctg aga acg tca acc cac cct gag 691 Tyr Val Ala Asp Gly AsnLeu Cys Leu Arg Thr Ser Thr His Pro Glu 185 190 195 tcc acc tgacaccccacac cttatttatg cgctgagccc tactccttcc 740 Ser Thr * 200 ttaatttatttcctctcacc ctttatttat gaagctgcag ccctgactga gacatagggc 800 tgagtttattgttttacttt tatacattat gcacaaataa acaacaagga attgga 856 7 200 PRT Homosapiens 7 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu GlyLeu 1 5 10 15 Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr ThrGly Lys 20 25 30 Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln GluLeu Ala 35 40 45 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu LysLeu Lys 50 55 60 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp AspLeu Arg 65 70 75 80 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu AlaGlu Leu Ala 85 90 95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro AlaLeu Glu Asp 100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu His His IleLeu Ser Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala GlyPro Arg Pro Arg Gly 130 135 140 Arg Leu His His Trp Leu His Arg Leu GlnGlu Ala Pro Lys Lys Glu 145 150 155 160 Ser Ala Gly Cys Leu Glu Ala SerVal Thr Phe Asn Leu Phe Arg Leu 165 170 175 Leu Thr Arg Asp Leu Lys TyrVal Ala Asp Gly Asn Leu Cys Leu Arg 180 185 190 Thr Ser Thr His Pro GluSer Thr 195 200 8 676 DNA Homo sapiens CDS (17)...(676) 8 gccctcggccaattgg atg gtg ccc acc aca ttg gca gag cca tgc cgc tgg 52 Met Val ProThr Thr Leu Ala Glu Pro Cys Arg Trp 1 5 10 gga agc agt tgc gat tta gccatg gct gca gct tgg acc gtg gtg ctg 100 Gly Ser Ser Cys Asp Leu Ala MetAla Ala Ala Trp Thr Val Val Leu 15 20 25 gtg act ttg gtg cta ggc ttg gccgtg gca ggc cct gtc ccc act tcc 148 Val Thr Leu Val Leu Gly Leu Ala ValAla Gly Pro Val Pro Thr Ser 30 35 40 aag ccc acc aca act ggg aag ggc tgccac att ggc agg ttc aaa tct 196 Lys Pro Thr Thr Thr Gly Lys Gly Cys HisIle Gly Arg Phe Lys Ser 45 50 55 60 ctg tca cca cag gag cta gcg agc ttcaag aag gcc agg gac gcc ttg 244 Leu Ser Pro Gln Glu Leu Ala Ser Phe LysLys Ala Arg Asp Ala Leu 65 70 75 gaa gag tca ctc aag ctg aaa aac tgg agttgc agc tct cct gtc ttc 292 Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser CysSer Ser Pro Val Phe 80 85 90 ccc ggg aat tgg gac ctg agg ctt ctc cag gtgagg gag cgc cct gtg 340 Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val ArgGlu Arg Pro Val 95 100 105 gcc ttg gag gct gag ctg gcc ctg acg ctg aaggtc ctg gag gcc gct 388 Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys ValLeu Glu Ala Ala 110 115 120 gct ggc cca gcc ctg gag gac gtc cta gac cagccc ctt cac acc ctg 436 Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln ProLeu His Thr Leu 125 130 135 140 cac cac atc ctc tcc cag ctc cag gcc tgtatc cag cct cag ccc aca 484 His His Ile Leu Ser Gln Leu Gln Ala Cys IleGln Pro Gln Pro Thr 145 150 155 gca ggg ccc agg ccc cgg ggc cgc ctc caccac tgg ctg cac cgg ctc 532 Ala Gly Pro Arg Pro Arg Gly Arg Leu His HisTrp Leu His Arg Leu 160 165 170 cag gag gcc ccc aaa aag gag tcc gct ggctgc ctg gag gca tct gtc 580 Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly CysLeu Glu Ala Ser Val 175 180 185 acc ttc aac ctc ttc cgc ctc ctc acg cgagac ctc aaa tat gtg gcc 628 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg AspLeu Lys Tyr Val Ala 190 195 200 gat ggg gac ctg tgt ctg aga acg tca acccac cct gag tcc acc tga 676 Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr HisPro Glu Ser Thr * 205 210 215 9 219 PRT Homo sapiens 9 Met Val Pro ThrThr Leu Ala Glu Pro Cys Arg Trp Gly Ser Ser Cys 1 5 10 15 Asp Leu AlaMet Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val 20 25 30 Leu Gly LeuAla Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr 35 40 45 Thr Gly LysGly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln 50 55 60 Glu Leu AlaSer Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu 65 70 75 80 Lys LeuLys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp 85 90 95 Asp LeuArg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala 100 105 110 GluLeu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala 115 120 125Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu 130 135140 Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg 145150 155 160 Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu AlaPro 165 170 175 Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr PheAsn Leu 180 185 190 Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala AspGly Asp Leu 195 200 205 Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr 210215 10 660 DNA Artificial Sequence degenerate sequence 10 atggtnccnacnacnytngc ngarccntgy mgntggggnw snwsntgyga yytngcnatg 60 gcngcngcntggacngtngt nytngtnacn ytngtnytng gnytngcngt ngcnggnccn 120 gtnccnacnwsnaarccnac nacnacnggn aarggntgyc ayathggnmg nttyaarwsn 180 ytnwsnccncargarytngc nwsnttyaar aargcnmgng aygcnytnga rgarwsnytn 240 aarytnaaraaytggwsntg ywsnwsnccn gtnttyccng gnaaytggga yytnmgnytn 300 ytncargtnmgngarmgncc ngtngcnytn gargcngary tngcnytnac nytnaargtn 360 ytngargcngcngcnggncc ngcnytngar gaygtnytng aycarccnyt ncayacnytn 420 caycayathytnwsncaryt ncargcntgy athcarccnc arccnacngc nggnccnmgn 480 ccnmgnggnmgnytncayca ytggytncay mgnytncarg argcnccnaa raargarwsn 540 gcnggntgyytngargcnws ngtnacntty aayytnttym gnytnytnac nmgngayytn 600 aartaygtngcngayggnga yytntgyytn mgnacnwsna cncayccnga rwsnacntrr 660 11 628 DNAHomo sapiens CDS (17)...(628) 11 gccctcggcc aattgg atg gtg ccc acc acattg gct tgg acc gtg gtg ctg 52 Met Val Pro Thr Thr Leu Ala Trp Thr ValVal Leu 1 5 10 gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct gtc cccact tcc 100 Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro Val Pro ThrSer 15 20 25 aag ccc acc aca act ggg aag ggc tgc cac att ggc agg ttc aaatct 148 Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser30 35 40 ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg gac gcc ttg196 Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu 4550 55 60 gaa gag tca ctc aag ctg aaa aac tgg agt tgc agc tct cct gtc ttc244 Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe 6570 75 ccc ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag cgc cct gtg292 Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val 8085 90 gcc ttg gag gct gag ctg gcc ctg acg ctg aag gtc ctg gag gcc gct340 Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala 95100 105 gct ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt cac acc ctg388 Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu 110115 120 cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc aca436 His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr 125130 135 140 gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg cac cggctc 484 Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu145 150 155 cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg gag gca tctgtc 532 Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val160 165 170 acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa tat gtggcc 580 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala175 180 185 gat ggg gac ctg tgt ctg aga acg tca acc cac cct gag tcc acctga 628 Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr *190 195 200 12 203 PRT Homo sapiens 12 Met Val Pro Thr Thr Leu Ala TrpThr Val Val Leu Val Thr Leu Val 1 5 10 15 Leu Gly Leu Ala Val Ala GlyPro Val Pro Thr Ser Lys Pro Thr Thr 20 25 30 Thr Gly Lys Gly Cys His IleGly Arg Phe Lys Ser Leu Ser Pro Gln 35 40 45 Glu Leu Ala Ser Phe Lys LysAla Arg Asp Ala Leu Glu Glu Ser Leu 50 55 60 Lys Leu Lys Asn Trp Ser CysSer Ser Pro Val Phe Pro Gly Asn Trp 65 70 75 80 Asp Leu Arg Leu Leu GlnVal Arg Glu Arg Pro Val Ala Leu Glu Ala 85 90 95 Glu Leu Ala Leu Thr LeuLys Val Leu Glu Ala Ala Ala Gly Pro Ala 100 105 110 Leu Glu Asp Val LeuAsp Gln Pro Leu His Thr Leu His His Ile Leu 115 120 125 Ser Gln Leu GlnAla Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg 130 135 140 Pro Arg GlyArg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro 145 150 155 160 LysLys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu 165 170 175Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu 180 185190 Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr 195 200 13 612 DNAArtificial Sequence degenerate sequence 13 atggtnccna cnacnytngcntggacngtn gtnytngtna cnytngtnyt nggnytngcn 60 gtngcnggnc cngtnccnacnwsnaarccn acnacnacng gnaarggntg ycayathggn 120 mgnttyaarw snytnwsnccncargarytn gcnwsnttya araargcnmg ngaygcnytn 180 gargarwsny tnaarytnaaraaytggwsn tgywsnwsnc cngtnttycc nggnaaytgg 240 gayytnmgny tnytncargtnmgngarmgn ccngtngcny tngargcnga rytngcnytn 300 acnytnaarg tnytngargcngcngcnggn ccngcnytng argaygtnyt ngaycarccn 360 ytncayacny tncaycayathytnwsncar ytncargcnt gyathcarcc ncarccnacn 420 gcnggnccnm gnccnmgnggnmgnytncay caytggytnc aymgnytnca rgargcnccn 480 aaraargarw sngcnggntgyytngargcn wsngtnacnt tyaayytntt ymgnytnytn 540 acnmgngayy tnaartaygtngcngayggn gayytntgyy tnmgnacnws nacncayccn 600 garwsnacnt rr 612 14 23DNA Artificial Sequence oligonucleotide ZC39270 14 aggcggaaga ggttgaaggtgac 23 15 22 DNA Artificial Sequence oligonucleotide ZC39272 15cgtcctagac cagccccttc ac 22 16 22 DNA Artificial Sequenceoligonucleotide ZC39271 16 gaccgtggtg ctggtgactt tg 22

What is claimed is:
 1. An isolated polypeptide comprising amino acidresidues 20 to 200 of SEQ ID NO:5.
 2. The isolated polypeptide of claim1 wherein the polypeptide comprises amino acid residues 1-200 of SEQ IDNO:5.
 3. The isolated polypeptide of claim 1 wherein the polypeptide hasantiviral activity to hepatitis B.
 4. The isolated polypeptide of claim1 wherein the polypeptide has antiviral activity to hepatitis C.
 5. Theisolated polypeptide of claim 1 wherein the polypeptide is a recombinantpolypeptide.
 6. The isolated poylpeptide of claim 5 wherein thepolypeptide is isolated from Escherichia coli.
 7. The isolatedpoylpeptide of claim 5 wherein the polypeptide is isolated from Chinesehamster ovary cells.
 8. An isolated polypeptide that has antiviralactivity and wherein nucleic acid molecule encoding the polypeptidehybridizes to SEQ ID NO:4 under washing conditions of 0.1×-0.2×SSC with0.1% SDS at 50-65° C.
 9. An isolated nucleic acid molecule comprisingnucleotides 58 to 600 of SEQ ID NO:4.
 10. The isolated nucleic acidmolecule of claim 9 comprising nucleotides 1-600 of SEQ ID NO:4.
 11. Anisolated nucleic acid molecule encoding a polypeptide wherein theencoded polypeptide comprises amino acid residues 20 to 200 of SEQ IDNO:5.
 12. The isolated nucleic acid molecule of claim 11 wherein theencoded polypeptide comprises amino acid residues 1 to 200 of SEQ IDNO:5.
 13. The isolated nucleic acid molecule of claim 11 wherein theencoded polypeptide has antiviral activity to hepatitis B.
 14. Theisolated nucleic acid molecule of claim 11 wherein the encodedpolypeptide has antiviral activity to hepatitis C.
 15. A vectorcomprising the isolated nucleic acid molecule of claim
 9. 16. Anexpression vector comprising the following operably linked elements: atranscription promoter; a nucleic acid molecule of claim 11; and atranscription terminator; and wherein the promoter is operably linkedwith the nucleic acid molecule; and wherein the nucleic acid molecule isoperably linked with the transcription terminator.
 17. A recombinanthost cell comprising the expression vector of claim 16, wherein the hostcell is selected from the group consisting of bacterium, yeast cell,fungal cell, insect cell, mammalian cell, and plant cell.
 18. Therecombinant host cell of claim 17 wherein the host cell is Escherichiacoli.
 19. The recombinant host cell of claim 17 wherein the host cell isa Chinese hamster ovary cell.
 20. A method of producing Zcyto21 protein,the method comprising: culturing recombinant host cells that comprisethe expression vector of claim 16, and that produce the Zcyto21 protein.21. The method of claim 20, further comprising isolating the Zcyto21protein from the cultured recombinant host cells.
 22. An antibody orantibody fragment that specifically binds with the polypeptide ofclaim
 1. 23. A formulation comprising: the polypeptide of claim 1; and apharmaceutically acceptable vehicle.